CN103461119A - Somatic embryogenesis and plant regeneration method for Picea asperata Mast - Google Patents
Somatic embryogenesis and plant regeneration method for Picea asperata Mast Download PDFInfo
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Abstract
The invention relates to a somatic embryogenesis and plant regeneration method for Picea asperata Mast. The method comprises the following steps: collecting freely-pollinated clonal cones of the Picea asperata Mast, and isolating immature zygotic embryos for somatic embryogenesis. The somatic embryogenesis comprises four phases: an embryonic callus induction phase, an embryonic callus proliferation phase, a somatic embryo mature phase and a somatic embryo germination phase. Compared with the conventional international research on the same or similar tree species, the frequency of embryonic callus induction is greatly increased, and the differentiation capacity and germination capacity of somatic embryos are also greatly increased. Meanwhile, a long-time keeping and proliferation method for embryonic callus is obtained, thus greatly reducing the risk of embryonic loss.
Description
Technical field
The present invention relates to cell engineering sapling multiplication field in forestry, particularly, relate to a kind of thick branch dragon spruce somatic embryo and occur and plant regeneration method.
Background technology
A thick branch dragon spruce (Picea asperata Mast) be Pinaceae, and the Picea seeds domesticly are commonly called as dragon spruce, also cry greatly really dragon spruce, are the widest Picea seeds of China's distribution.Aiphyllium, up to 45m, the diameter of a cross-section of a tree trunk 1.3 meters above the ground reaches 1m.The bark ficelle, be cleaved into irregular scale or slightly thick fast sheet comes off; Sprig has the pubescence of dredging living or close life, or without hair, yellow, the isabelline or light red brown of light brown when annual, pulvinus has white powder, or white powder is not obvious; The hibernaculum taper shape, have resin, and base portion expands.Needle four rib shape bar shapeds, long 1～2cm, wide 1～1.5mm, microbend, the micro-point of tip or anxious point, cross section four prismatics.The cylindric square of cone is circular or cylindrical, and upper end is gradually narrow, ripe front green, filbert or Chestnut when ripe, long 5～16cm, footpath 2.5～3.5cm; Middle part fruit scale obovate; Seed falls oval, is about 4mm, and fructus forsythiae is about 1.5cm, and seed wing is filbert, the shape of falling ovum square circle; 6～7 pieces of cotyledons.4～May of florescence, cone maturation in 9～October.For China endemic species, mainly be distributed in the high Mountain area in Asia in Chuan Xi plateau, the southeast of Gansu and the west and south, Shaanxi, mean sea level is distributed in the high mountain hilly country of 2400～3600m.Thick branch dragon spruce is the shallow root seeds, slightly anti-the moon, ability drying and cold environment.The timber yellow-white, more light and soft, texture is straight, and structure is thin, flexible.Can make the material such as papermaking, building, furniture and the xylem fibre raw material of industry.Material is good, and growth is fast, and strong adaptability, be the Major Tree Species Planted of area.At present, artificial afforestration is with seedling mainly by the breeding of growing directly from seeds, but thick branch dragon spruce solid evening, the seed maturity rate is low, and, with serious " biennial bearing " phenomenon, supply falls short of demand to cause thick branch dragon spruce good seed.
The plant somatocyte embryo generation technique, refer in vitro, and the somatic cell broken up, under specific environmental condition, develops into the process with the similar embryoid of zygotic embryo.Plant somatocyte embryo is to utilize the cell engineering means to realize one of important channel of plant regeneration, as a kind of, utilize artificial means can realize to greatest extent that plant expands numerous measure, especially for being difficult to longer coniferous tree seeds of cottage propagation and breeding cycle, there is huge using value.Simultaneously, because somatic embryo and zygotic embryo have similar morphosis and growth course, Somatic Embryogenesis has again relative genetic stability; Carry out the research that plant somatocyte embryo occurs, for the genesis mechanism research that discloses plant embryos with carry out Study on Genetic Transformation a kind of model study system is provided.
Coniferous species is one of the class seeds the most widely that distribute in world wide, owing to usually having good wood property, as developed country and regional of paramount importance industrial cut stock seeds such as North America and Northern Europe.Yet, because it is different from phanerogamic characteristic, coniferous species is more responsive to isolated condition, be class seeds of difficulty maximum in somatic embryo generation research process.According to incompletely statistics, up to now, the whole world more than 150 seeds of having an appointment have been realized the somatic embryo generation, and wherein coniferous species is only had an appointment 50 kinds and realized that somatic embryo occurs and plant regeneration.
At present, about thick branch dragon spruce somatic embryo, occur and plant regeneration method, there is no bibliographical information both at home and abroad.Therefore, develop an a kind of efficiently thick branch dragon spruce somatic embryo and occur and plant regeneration method, for the factorial seedling growth based theoretical that realizes thick branch dragon spruce somatic embryo with provide particularly necessary practicable method; Simultaneously, also can set up good experimental system for biotechnology breeding, germ plasm resource preservation, Fast-propagation, the genetic transformation of thick branch dragon spruce.
Summary of the invention
In order to solve the problems of the prior art, the purpose of this invention is to provide a kind of thick branch dragon spruce somatic embryo and occur and plant regeneration method.
A kind of thick branch dragon spruce somatic embryo provided by the invention occurs and plant regeneration method, comprising: gather the clonal cone of thick branch dragon spruce open pollination, isolate immature zygotic embryos (carrying out the selection of explant) 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, in the selection of described explant, adopting the immature zygotic embryo of growth of thick branch dragon spruce open pollination is material, and it is the last ten-days period in mid-June to September that cone gathers the date, preferably the middle ten days and the last ten days in June.Isolate immature zygotic embryos and carry out inducing of embryo callus, whole Induction Process carries out under aseptic condition.
Wherein, the described embryonic callus induction stage: adopt the 1/2LM minimal medium, additional auximone 2,4-Benzene Chloride fluoroacetic acid (2,4-dichlorophenoxyacetic acid, hereinafter to be referred as 2, concentration 4-D) is 5～15 μ M, the concentration of basic element of cell division 6-benzylaminopurine (benzylaminmopurine, hereinafter to be referred as 6-BA) is 2.5～10 μ M.Described 2,4-D is 10 μ M preferably, and 6-BA is 5 μ M preferably.
Further, in embryonic callus induction stage 1/2LM minimal medium used, also comprise: the sucrose that concentration is 1%, 500mgL
-1glutamine, 1gL
-1casein hydrolysis and 0.2% plant gel, can obtain the highest embryonic callus induction frequency under this condition.
Wherein, the described embryo callus multiplicative stage: adopt the 1/2LM liquid nutrient medium, additional hormone is 2 of 3.75～10 μ M, the 6-BA of 4-D and 1.25～7.5 μ M, preferably 2 of 3.75～5 μ M, the 6-BA of 4-D and 1.25～3.75 μ M, more preferably 2 of 5 μ M, the 6-BA of 4-D and 3.75 μ M, this condition can obtain the best hormone combinations of embryo callus propagation, maintain the height embryo of embryo callus, obtain higher embryo callus harvest yield simultaneously.
Wherein, the 1/2LM liquid nutrient medium that the described embryo callus multiplicative stage is used, cell volume and culture fluid volume ratio are 1:3～1:15, preferably 1:5～1:15, more preferably 1:7; When being 1:7, volume ratio can obtain maximum embryo callus harvest yield.
Further, in embryonic callus induction stage 1/2LM minimal medium (being inducing culture) used, the concentration of 2,4-D is preferably 2 times of 6-BA; In 1/2LM liquid nutrient medium (being proliferated culture medium) used of described embryo callus multiplicative stage, 2, the concentration of a little higher than 6-BA of concentration of 4-D, and in proliferated culture medium, hormone concentration obviously reduces than inducing culture, and this kind of hormone combinations is conducive to the maintenance of the callus of height embryo.
Wherein, in 1/2LM liquid nutrient medium used of described embryo callus multiplicative stage, adopt sucrose as carbon source material, concentration is 0.5～3.0%, preferably 1～1.5%, most preferably 1%(is consistent with sucrose concentration in inducing culture most preferably the time).
Wherein, in 1/2LM liquid nutrient medium (being proliferated culture medium) used of described embryo callus multiplicative stage, very crucial is exactly a bit to reduce to greatest extent the cell contamination frequency suspended while cultivating.
In the present invention, embryo callus is placed in the conical flask of the 250ml that liquid nutrient medium is housed and cultivates, before cultivating, need the conical flask autoclaving, after sealing with the ventilation sealed membrane after inoculation, need the aseptic newspaper of sealing two layers again, can greatly reduce the risk of cell contamination like this, make Contamination rate control in 1%.
Wherein, in 1/2LM liquid nutrient medium used of described embryo callus multiplicative stage, also comprise: 500mgL
-1glutamine, 1gL
-1casein hydrolysis.Breed while cultivating, shaking speed is 110 rev/mins.
The method of the invention, the hormone concentration that the embryonic callus induction stage adopts can obtain high embryonic callus induction frequency.In the propagation of embryo callus is cultivated, what the present invention adopted is the liquid suspension training method, and condition of culture is optimized, and can greatly maintain the embryo sexuality of embryo callus, reduces the risk that it loses the embryo sexuality; Simultaneously, can obtain embryo callus a large amount of and the height homogenization, be conducive to the differentiation of later stage high level of synchronization somatic embryo.
Wherein, the described somatic embryo stage of ripeness: adopt the 1/2LM minimal medium, the abscisic acid that additional hormone is 31～123 μ M (abscisic acid, hereinafter to be referred as ABA), preferred 62～123 μ M, more preferably 92～123 μ M, further preferred 100～108 μ M; Under the ABA of 100 μ M processes, can obtain a large amount of and the higher mature somatic embryo of synchronization degree, cost is calculated the sprouting that is conducive to again the later stage mature somatic embryo like this.
Wherein, in middle 1/2LM minimal medium used of the described somatic embryo stage of ripeness, also comprise: osmotic adjustment Macrogol 4000 (being called for short PEG4000), concentration is 0～7.5%, preferably 3.5～7.5%, more preferably 5～7.5%, more preferably 5%.
Wherein, in middle 1/2LM minimal medium used of the described somatic embryo stage of ripeness, adopt 1～9% sucrose, preferably 1～4.5%, more preferably 1～3%, further preferably 3%.
Wherein, in middle 1/2LM minimal medium used of the described somatic embryo stage of ripeness, adopt 0～0.5% active carbon, preferably 0.1～0.5%, more preferably 0.1%.
Wherein, in middle 1/2LM minimal medium used of the described somatic embryo stage of ripeness, adopt 0.2～0.8% plant gel, preferably 0.2～0.6%, more preferably 0.4%.
Wherein, in middle 1/2LM minimal medium used of the described somatic embryo stage of ripeness, also comprise: 500mgL
-1glutamine and 1gL
-1caseinhydrolysate.
Wherein, the condition of culture in described embryonic callus induction stage, embryo callus multiplicative stage and the somatic embryo stage of ripeness can be all: add before gel pH is adjusted to 5.8, sterilizing 20 minutes under 121 ℃, pressure 101kp high temperature, condition of high voltage subsequently; 24 ± 1 ℃ of cultivation temperature, dark condition is cultivated.
The method of the invention, by on affecting somatic embryo inducement and ripe many factors is tested, the best maturation medium combination that screening obtains, be conducive to realize that a large amount of and high synchronization of thick branch dragon spruce somatic embryo produces, be conducive to induce the somatic embryo maturation of generation, for the realization of thick branch dragon spruce factorial seedling growth provides the most key assurance.
Wherein, described somatic embryo is sprouted the stage: adopt the 1/4LM minimal medium, concentration of activated carbon is 0～0.5%, preferably 0.1～0.4%, more preferably 0.2%.
Wherein, described somatic embryo is sprouted stage 1/4LM minimal medium used, and adopting drying time is 0～21 day, preferred 1-10 days, more preferably 4～10 days, most preferably 4 days.
Wherein, described somatic embryo is sprouted in stage 1/4LM minimal medium used, adopts 0～15% PEG4000, and preferably 0～10%, more preferably 0%, do not add the sprouting that PEG4000 is conducive to somatic embryo.
Wherein, described somatic embryo is sprouted in stage 1/4LM minimal medium used, adopts 1～15% sucrose, and preferably 1～7%, more preferably 1～4%, most preferably 2%.
Wherein, described somatic embryo is sprouted in the medium in stage, also comprises: 500mgL
-1glutamine, 1gL
-1caseinhydrolysate and 0.4% plant gel.
Wherein, in the germination process of described somatic embryo, somatic embryo is placed horizontally to the semisolid culturemedium surface, culture dish inclination 45° angle is placed, and is conducive to cotyledon stretching, extension, plumular axis and radicle elongation; Simultaneously, the radicle of growth is attached to media surface, and while having avoided transplanting, the tender shoot root of children is impaired, is conducive to transplant survival.
Wherein, the condition of culture in the sprouting stage of described somatic embryo is: cultivation temperature is 24 ± 1 ℃; First week (take 7 days as the cycle), dark culturing; Second week (take 7 days as the cycle), 20 μ molm
-2s
-1under illumination, and hide with one deck paper (newspaper), the photoperiod is 16h illumination and 8h dark; Subsequently, 50 μ molm
-2s
-1under illumination, the photoperiod is 16h illumination and 8h dark culturing.
Wherein, in the medium in each stage of the present invention, due to the glutamine and the ABA non-refractory that use, adopt the mode of filtration sterilization, other all the components all adopt the sterilizing of HTHP mode.
Method of the present invention, before immature zygotic embryos is carried out to embryonic callus induction, also comprises: the prematurity cone is carried out disinfection, then under aseptic environment, take out immature zygotic embryos.
Wherein, described disinfection way is: utilize absolute ethyl alcohol to thick branch dragon spruce prematurity cone (being the clonal cone of the above-mentioned thick branch dragon spruce open pollination) 15～20min that sterilizes, then with aseptic water washing at least 2 times.This kind of disinfection way can make Contamination rate control in 0.1%, can meet the needs of practical operation fully, and simple to operate, efficient, cost is low, environmental pollution is little.
The percent concentration occurred in the present invention " % " all refers to that quality-volumetric concentration g/100mL(is the grams that needs the material of interpolation in 100mL solution).
Up to now, the external tender stem apex of children that adopts is that explant is induced at most and can only be induced embryo callus, all can not be divided into somatic embryo.Cotyledon, plumular axis and radicle that the present inventor attempted with seed sprouting are that explant is induced, and can induce callus, but frequency of embryonic callus induction are extremely low, and can not continue normal propagation, can not carry out the differentiation of somatic embryo.So the present invention utilizes whole zygotic embryo as explant, and the explant of development in different stages is induced to contrast, screening obtains optimum immature zygotic embryos developmental stage, meets the needs of research and actual production.
In addition, the application, by a large amount of experiments, filters out for the somatic embryo generation of thick branch dragon spruce and the specified conditions of plant regeneration, is conducive to improve the induction frequency of embryo callus; Be conducive to maintain the embryo sexuality of embryo callus, reduced the risk of losing the embryo sexuality; Be conducive to realize a large amount of and high synchronized acquisition of somatic embryo; Be conducive to improve the germination efficiency of somatic embryo.
Method provided by the invention, specially low for the natural ripening rate of thick branch dragon spruce and there is serious " biennial bearing " phenomenon, the cuttage root-taking difficulty, the sapling multiplication technology can't meet the present situation of large tracts of land afforestation needs, finds a kind of somatic embryo generation of thick branch dragon spruce choiceness and the technology of plant regeneration.With existing, belong to together in the world or the research report of similar seeds is compared, frequency of embryonic callus induction greatly improves, the differentiation capability of somatic embryo and sprouting ability also greatly improve, screening simultaneously obtains keeping for a long time and enrichment procedure of embryo callus, has greatly reduced the risk of losing embryo.Research maintains the leading position, for the factorial seedling growth based theoretical that realizes thick branch dragon spruce somatic embryo with provide particularly necessary practicable method; Simultaneously, also can set up good experimental system for biotechnology breeding, germ plasm resource preservation, Fast-propagation, the genetic transformation of thick branch dragon spruce.
The accompanying drawing explanation
The immature zygotic embryos that Fig. 1 is thick branch dragon spruce.
The embryo callus that Fig. 2 is thick branch dragon spruce.
The mature somatic embryo that Fig. 3 is thick branch dragon spruce.
The plant that the somatic embryo that Fig. 4 is thick branch dragon spruce is sprouted.
Embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.
The present invention plant gel gellan used gum, adopt the Sigma brand.It is a kind of epoxy resin, identical with the effect of agar, but effect is well a lot, and the rate of set and the transparency that are embodied in medium are improved.Active carbon activated carbon used in the present invention, adopt the Sigma brand equally, with the active carbon of selling on open market, compares, and its activated adoption ability is stronger.Also can use on market and can purchase available conventional reagent.
Water used in the present invention is deionized water, and purity is 18.2 megaohms.The present invention's other raw materials used and reagent are the conventional reagent of buying from the market.
The composition of the present invention's LM minimal medium used is:
Macroelement: 1.65gL
-1nH
4nO
3, 1.9gL
-1kNO
3, 1.85gL
-1mgSO
47H
2o, 0.34gL
-1kH
2pO
4, 0.022gL
-1caCl
22H
2o.
Trace element: 0.0304gL
-1h
3bO
3, 0.043gL
-1znSO
47H
2o, 0.021gL
-1mnSO
4h
2o, 0.00126gL
-1na
2moO
42H
2o, 0.00415gL
-1kI, 0.0005gL
-1cuSO
45H
2o, 0.000125gL
-1coCl
26H
2o.
Molysite: 0.0278gL
-1feSO
47H
2o, 0.0373gL
-1eDTA2H
2the O(ethylenediamine tetra-acetic acid).
Inositol: 0.1gL
-1myo-Inositol.
Organic salt: 0.0001gL
-1thiamine-HCl(Vitamin B
1), 0.0005gL
-1niacin acid(Vitamin B
3), 0.0001gL
-1pyroxidone-HCl(Vitamin B
6)
The 1/2LM minimal medium that the present invention occurs, be exactly the composition to above-mentioned LM minimal medium, and macroelement, trace element and molysite are reduced by half, and other are constant.The 1/4LM minimal medium that the present invention occurs, be exactly to above-mentioned LM minimal medium, and macroelement, trace element and molysite are reduced to 1/4, and other are constant.The 1/2LM liquid nutrient medium that the present invention occurs, be exactly to above-mentioned LM minimal medium, and macroelement, trace element and molysite are reduced by half, and other are constant, do not add gel in medium simultaneously.The 1/2LM semisolid culturemedium that the present invention occurs, be exactly to above-mentioned LM minimal medium, and macroelement, trace element and molysite are reduced by half, and other are constant, adds gel in medium simultaneously.
Wherein, first prepare mother liquor during actual the use, then dilution is used.The mother liquor of macroelement and molysite is mixed with to 100 times, and trace element is mixed with 1000 times, and inositol and organic salt are mixed with 500 times.For fear of CaCl in macroelement
22H
2o forms precipitation with other compositions combinations, by its independent preparation.
Content of the present invention is the comprehensive of test data between 2011～2013 years, and experimental result is stable, reliable.Described experiment material is picked up from the national germplasm resource bank of little Gansu Province, Gansu Province mountain forest industry pilot office forestry research institute dragon spruce.
Isolate immature zygotic embryos: gather the prematurity cone that left and right open pollination on May 1 produces, start to carry out the collection of prematurity cone at the beginning of 6 months, gathered once every 7～10 days, gather altogether 15 times, 12 genotype, place the low temperature under 4 ℃ of conditions of the cone under adopting.Process cone 15min, aseptic water washing 2 times with absolute ethyl alcohol.Under aseptic condition, take out seed and peel off kind of skin and endosperm, immature zygotic embryos (as shown in Figure 1) is carried out to inducing of embryo callus.
Embodiment 2
The embryonic callus induction stage: the immature zygotic embryos that embodiment 1 is obtained carries out embryonic callus induction, adopts the 1/2LM minimal medium, and additional hormone concentration is as shown in the table respectively:
| Processing number | 2,4-D/μM | 6- |
| 1 | 5 | 2.5 |
| 2 | 5 | 5 |
| 3 | 5 | 10 |
| 4 | 10 | 2.5 |
| 5 | 10 | 5 |
| 6 | 10 | 10 |
| 7 | 15 | 2.5 |
| 8 | 15 | 5 |
| 9 | 15 | 10 |
And, add 1gL
-1the natural complex casein hydrolysis, in medium containing 1% sucrose, 500mgL
-1glutamine and 0.2% plant gel (gellan gum, Sigma).Add before gel pH is adjusted to 5.8, sterilizing 20 minutes under 121 ℃, pressure 101kp high temperature, condition of high voltage subsequently.Fill about 35ml semisolid culturemedium in the glass culture dish of diameter 90mm.24 ± 1 ℃ of cultivation temperature, dark condition is cultivated, to inducing embryo callus.
Immature zygotic embryos was inoculated in callus inducing medium after 2-3 days, and zygotic embryo starts to start, and was embodied in embryo and expanded, and the plumular axis base portion has the brown projection to occur, cotyledon differentiates " spot-like projections "; After 1 week, whole zygotic embryo changes callus into; After 2 weeks, embryo callus starts to occur, embryo callus subculture propagation is organized gradually and grown up together with non-embryonic callus subsequently, and 6-7 can separately breed embryo callus separately after week.The non-embryonic callus tissue is many to be differentiated by plumular axis, and fast, majority is the brown graininess in growth, and differentiation phase can not produce normal somatic embryo; The embryo callus majority is transformed by " spot-like projections " of cotyledon differentiation, is embodied in white, transparent, thickness, thread, and possesses the ability that differentiates normotrophic somatic embryo.
Immature zygotic embryos with mid-June to late June can obtain the highest embryonic callus induction frequency, and average Induce of embryoid rate is more than 60%, and high Induce of embryoid genotype can reach more than 95%.Now, zygotic embryo was grown in the early stage cotyledonary embryos stage.With 2 of 10 μ M, under the 6-BA of 4-D and 5 μ M processes (as following embodiment 3), the frequency of embryonic callus induction of acquisition is the highest, and the combination of taking second place is 2,4-D15 μ M and 6-BA5 μ M, and the poorest combination is 2,4-D5 μ M and 6-BA10 μ M; Hormone concentration is too high or too low all is unfavorable for inducing of embryo callus.
Embodiment 3
The embryonic callus induction stage: the immature zygotic embryos that embodiment 1 is obtained carries out embryonic callus induction, adopts the 1/2LM minimal medium, and additional hormone concentration is respectively 2,4-D10 μ M and 6-BA5 μ M, adds 1gL
-1the natural complex casein hydrolysis, in medium containing 1% sucrose, 500mgL
-1glutamine and 0.2% plant gel.Add before gel pH is adjusted to 5.8, sterilizing 20 minutes under 121 ℃, pressure 101kp high temperature, condition of high voltage subsequently, 24 ± 1 ℃ of cultivation temperature, dark condition is cultivated, to inducing embryo callus.Under the best collection date, a plurality of genotypic average inductivities reach more than 64%.
Embodiment 4
The multiplicative stage of embryo callus: the embryo callus that embodiment 3 is obtained is bred, use be the 1/2LM semisolid culturemedium, growth hormone 2,4-D concentration is 10 μ M, basic element of cell division 6-BA concentration is 5 μ M, sucrose concentration is 1%.Casein hydrolysis concentration is 1gL
-1, glutamine is 500mgL
-1, gel strength is 0.2%.Condition of culture is all identical with induction period.Place the embryo callus of 7 diameter 0.5cm sizes in each culture dish (diameter 90mm), within two weeks, transfer once, during switching, the embryo callus amount of growth can double manyly, and monolithic diameter 1～1.5cm(as shown in Figure 2).
Embodiment 5
The multiplicative stage of embryo callus: the embryo callus that embodiment 4 is obtained is got about 1g.Be placed in the 250ml conical flask, place the 50ml liquid nutrient medium in bottle, carry out liquid suspension propagation.The 1/2LM minimal medium, do not add gel.2,4-D concentration is 10 μ M, and 6-BA concentration is 5 μ M, and sucrose concentration is 1%.Casein hydrolysis concentration is 1gL
-1, glutamine is 500mgL
-1, shaking speed is 110 rev/mins, 24 ± 1 ℃ of cultivation temperature, and dark condition is cultivated.After 10 days, by the embryo callus switching, the free callus that during switching, just switching shakes up, the switching medium is identical; So, obtain the suspension cell line of embryo callus after 1 month.
Embryo callus is placed in the conical flask of the 250ml that liquid nutrient medium is housed and cultivates, before cultivating, need the conical flask autoclaving, after sealing with the ventilation sealed membrane after inoculation, need the aseptic newspaper of sealing two layers again, can greatly reduce the risk of cell contamination like this, make Contamination rate control in 1%.
Embodiment 6
The multiplicative stage of embryo callus: the embryo callus suspension cell with acquisition in embodiment 5 is material, cell volume (standing 1h) is set and with the ratio (inoculum concentration) of culture fluid, is respectively 1:5,1:7,1:9,1:12 and 1:15.The 50ml liquid nutrient medium is placed in the 250ml conical flask and cultivates, and medium component and condition of culture are with embodiment 5.Continue to cultivate after 9 days, the amount of growth of embryo callus is as shown in the table:
| Inoculum concentration (V:V) | 1:5 | 1:7 | 1:9 | 1:12 | 1:15 |
| Amount of growth (g/100ml) | 20.348±6.161 | 24.696±4.083 | 20.420±2.652 | 16.274±3.842 | 20.463±6.065 |
As can be seen from the above table: when cell volume and culture fluid volume ratio are 1:7, can obtain maximum embryo callus amount of growth, suspend propagation after 9 days, can obtain the fresh weight of 24.696g/100ml, while rigidly connecting kind, increase by 4.24 times.
Embodiment 7
The multiplicative stage of embryo callus: the embryo callus suspension cell with acquisition in embodiment 5 is material, it is 1:7 with the ratio of culture fluid that cell volume (standing 1h) is set, reset growth hormone 2, the concentration of 4-D is respectively 3.75,5,7.5 and 10 μ M, and 6-BA concentration is fixed as 5 μ M.The 70ml liquid nutrient medium is placed in the 250ml conical flask and cultivates, and other medium components and condition of culture are with embodiment 5.Continue to cultivate after 9 days, the amount of growth of embryo callus is as shown in the table:
| 2,4-D concentration (μ M) | 3.75 | 5 | 7.5 | 10 |
| Amount of growth (g/100ml) | 23.463±3.299 | 24.034±6.596 | 18.241±2.420 | 12.136±1.889 |
As can be seen from the above table, along with growth hormone 2, the raising of 4-D concentration, the growth of embryo callus presents the trend that existing increase reduces afterwards.When 2,4-D concentration is 5 μ M, can obtain maximum embryo callus amount of growth, suspend propagation after 9 days, can obtain the fresh weight of 24.034g/100ml, increased by 4.54 times while rigidly connecting kind.
Embodiment 8
The multiplicative stage of embryo callus: the embryo callus suspension cell with acquisition in embodiment 5 is material, it is 1:7 with the ratio of culture fluid that cell volume (standing 1h) is set, the concentration that resets basic element of cell division 6-BA is respectively 1.25,2.5,3.75,5 and 7.5 μ M, 2,4-D concentration is fixed as 5 μ M.The 70ml liquid nutrient medium is placed in the 250ml conical flask and cultivates, and other medium components and condition of culture are with embodiment 5.Continue to cultivate after 9 days, the amount of growth of embryo callus is as shown in the table:
| 6-BA concentration (μ M) | 1.25 | 2.5 | 3.75 | 5 | 7.5 |
| Amount of growth (g/100ml) | 13.110±3.056 | 12.249±1.628 | 13.571±3.179 | 10.583±3.800 | 11.354±3.748 |
As can be seen from the above table, when 6-BA concentration is 3.75 μ M, can obtain maximum embryo callus amount of growth, suspend propagation after 9 days, can obtain the fresh weight of 13.571g/100ml, while rigidly connecting kind, increase by 4.06 times.And, in the multiplicative stage of embryo callus, additional hormone is 2 of 5 μ M, the 6-BA of 4-D and 3.75 μ M, the effect optimum, be the best hormone combinations that can obtain embryo callus propagation.
Embodiment 9
The multiplicative stage of embryo callus: the embryo callus suspension cell with acquisition in embodiment 5 is material, and cell volume (standing 1h) is 1:7 with the ratio of culture fluid.Reset sucrose concentration and be respectively 0.5%, 1%, 1.5%, 2% and 3%.The concentration of 2,4-D is 5 μ M, and 6-BA concentration is 5 μ M.The 70ml liquid nutrient medium is placed in the 250ml conical flask and cultivates, and other medium components and condition of culture are with embodiment 5.Continue to cultivate after 9 days, the amount of growth of embryo callus is as shown in the table:
| Sucrose concentration (%) | 0.5 | 1 | 1.5 | 2 | 3 |
| Amount of growth (g/100ml) | 12.638±1.358 | 15.566±0.147 | 13.595±2.340 | 12.602±2.693 | 12.888±2.374 |
As can be seen from the above table, when sucrose concentration is 1%, can obtain maximum embryo callus amount of growth, suspend propagation after 9 days, can obtain the fresh weight of 15.566g/100ml, while rigidly connecting kind, increase by 4.44 times.
Annotate: the embryo callus suspension cell of the initial inoculation that embodiment 6～9 is used is material, and fresh weight separately is different, with the actual fresh weight used of each embodiment, is as the criterion.
Embodiment 10
The stage of ripeness of somatic embryo: the embryo callus that embodiment 4 is obtained carries out the maturation of somatic embryo to be cultivated, and adopts the 1/2LM minimal medium.ABA concentration is set and is respectively 31,46,62 and 92 μ M, PEG4000 concentration is respectively 0%, 2.5%, 5% and 7.5%, and sucrose concentration is respectively 1%, 3%, 6% and 9%, and concentration of activated carbon is respectively 0,0.1%, 0.3% and 0.5%, gel strength is respectively 0.2%, 0.4%, 0.6% and 0.8%, adopts L
16(4
5) method carries out Orthogonal Composite.In addition, in medium, caseinhydrolysate substitutes the casein hydrolysis of inducing with the multiplicative stage, and concentration is 1gL
-1, the concentration of glutamine is 500mgL
-1, add before gel pH be adjusted to 5.8, sterilizing 20 minutes under 121 ℃, pressure 101kp subsequently.Differentiation phase, tiling one deck Whatman filter paper in differential medium, under aseptic condition, tile the embryo callus of about 1g left and right on filter paper, notices that tiling evenly.24 ± 1 ℃ of lower dark culturing.
Embryo callus was seeded in differential medium after 2 days, and globular embryo starts to occur, globular embryo quantity increases subsequently; Within 7 days, with the rear section globular embryo, change torpedo-shape embryo into, torpedo-shape embryo quantity continues to increase subsequently; 3 weeks start to enter the cotyledonary embryos state with the rear section globular embryo, and after 5 weeks, cotyledonary embryos is obvious, enters successively maturity state.Ripe somatic embryo stalwartness, plumular axis is obvious, and whole somatic embryo is faint yellow.Each factor variable concentrations is processed the lower mature somatic embryo situation obtained and is seen the following form:
| ABA concentration (μ M) | 31 | 46 | 62 | 92 |
| Mature somatic embryo quantity (individual/gram) | 59 | 40 | 71 | 138 |
| PEG4000 concentration (%) | 0 | 2.5 | 5 | 7.5 |
| Mature somatic embryo quantity (individual/gram) | 16 | 85 | 122 | 117 |
| Sucrose concentration (%) | 1 | 3 | 6 | 9 |
| Mature somatic embryo quantity (individual/gram) | 155 | 168 | 16 | 1 |
| Concentration of activated carbon (%) | 0 | 0.1 | 0.3 | 0.5 |
| Mature somatic embryo quantity (individual/gram) | 50 | 105 | 84 | 100 |
| Gel strength (%) | 0.2 | 0.4 | 0.6 | 0.8 |
| Mature somatic embryo quantity (individual/gram) | 66 | 133 | 84 | 57 |
As seen from the above table, in form, the numerical value of each condition can combination collocation, thereby carries out the setting of each condition in stage of ripeness of somatic embryo.In the present embodiment, best realignment and regrouping is ABA92 μ M, PEG40005%, sucrose 3%, active carbon 0.1% and gel 0.4%.
Embodiment 11
The stage of ripeness of somatic embryo: the embryo callus that embodiment 4 is obtained carries out the maturation of somatic embryo and cultivates, adopt the 1/2LM minimal medium, ABA concentration is set and is respectively 62,77,85,92,100,108 and 123 μ M, fixing additional PEG4000 concentration is 5%, sucrose concentration is 3%, concentration of activated carbon is 0.1%, and gel strength is 0.4%.Other medium components and condition of culture are with embodiment 10.The differentiated result of somatic embryo sees the following form:
| ABA concentration (μ M) | 62 | 77 | 85 | 92 | 100 | 108 | 123 |
| Mature somatic embryo quantity (individual/gram) | 310 | 218 | 81 | 206 | 516 | 583 | 137 |
As seen from the above table, when ABA concentration is 100～108 μ M, the mature somatic embryo quantity that every gram embryo callus obtains is maximum.The somatic embryo synchronization degree obtained under 100 μ M and 108 μ M ABA conditions is all very high, and variance analysis shows that the quantity variance of somatic embryo of the two acquisition is not remarkable.Consider in addition the simplicity (100 μ M easily convert and preparation) of Financial cost and practical operation, finally determine the optium concentration that 100 μ M ABA are embryo differentiate, can obtain somatic embryo quantity is 516/gram; Somatic embryo in the performance of sprouting stage better in addition.
Embodiment 12
The stage of ripeness of somatic embryo: the embryo callus that embodiment 4 is obtained carries out the maturation of somatic embryo and cultivates, adopt the 1/2LM minimal medium, PEG4000 concentration is set and is respectively 4.5%, 5%, 5.5%, 6.5% and 7.5%, fixing additional ABA92 μ M, other medium components and condition of culture are with embodiment 11.The differentiated result of somatic embryo sees the following form:
| PEG4000 concentration (%) | 4.5 | 5 | 5.5 | 6.5 | 7.5 |
| Mature somatic embryo quantity (individual/gram) | 62 | 206 | 84 | 70 | 86 |
As seen from the above table, when PEG4000 concentration is 5%, the mature somatic embryo quantity that every gram embryo callus obtains is maximum.
It should be noted that embryo callus, before differentiation, need to not carry out preculture and can normally break up on the medium that does not add hormone, can on the basis used manpower and material resources sparingly, shorten cultivation cycle.
Embodiment 13
The stage of ripeness of somatic embryo: the embryo callus that embodiment 4 is obtained carries out the maturation of somatic embryo to be cultivated, and adopts the 1/2LM minimal medium, adopts ABA100 μ M, PEG40005%, sucrose 3%, active carbon 0.1% and gel 0.4%, other medium components and condition of culture are with embodiment 11.Differentiation produces a large amount of and high synchronized somatic embryo (as shown in Figure 3), and mature somatic embryo quantity (individual/gram) is 516/gram.
Embodiment 14
The stage of ripeness of somatic embryo: using the optimum condition that obtains separately in embodiment 6～9 as liquid suspension proliferated culture medium condition, adopt the 1/2LM minimal medium, the ratio that cell volume and culture fluid are set is 1:7, additional 5 μ M2,4-D, 3.75 the sucrose of μ M6-BA and 1%, other medium components and condition of culture are with embodiment 5.Cultivate after 9 days, embryo callus is carried out to the maturation of somatic embryo and cultivate.Somatic embryo maturation medium composition and condition of culture are with embodiment 11.This kind of mode be because the embryo callus developmental condition is consistent, the quality homogeneous; Not only reduced embryo callus and lost the risk of embryo, can obtain quantitatively and somatic embryo preferably all qualitatively simultaneously, the sprouting that meets the later stage mature somatic embryo is processed.
Embodiment 15
The sprouting stage of somatic embryo: the somatic embryo that embodiment 13 is obtained is sprouted, and uses the 1/4LM minimal medium, sucrose concentration is set and is respectively 1%, 2%, 4%, 7%, 10% and 15%, does not add any hormone in medium, and additional glutamine 500mgL
-1, 1gL
-1caseinhydrolysate, active carbon 0.1% and 0.4% plant gel.Cultivation temperature is 24 ± 1 ℃.First week, dark culturing; Second week, 20 μ molm
-2s
-1under illumination, and hide with one deck newspaper, the photoperiod is 16h illumination and 8h dark; Subsequently, 50 μ molm
-2s
-1under illumination, the photoperiod is 16h illumination and 8h dark culturing.
In the germination process of somatic embryo, somatic embryo is placed horizontally to the semisolid culturemedium surface, culture dish inclination 45° angle is placed, and is conducive to cotyledon stretching, extension, plumular axis and radicle elongation; Simultaneously, the radicle of growth is attached to media surface, and while having avoided transplanting, the tender shoot root of children is impaired, is conducive to transplant survival.
Sprout and the results are shown in following table after three weeks:
| Sucrose concentration (%) | 1 | 2 | 4 | 7 | 10 | 15 |
| Germination rate (%) | 16 | 25 | 17 | 3 | 0 | 0 |
As seen from the above table, when sucrose concentration is 2%, the germination rate of somatic embryo is the highest, can reach 25%, the too low and too high sprouting that all is unfavorable for somatic embryo of sucrose concentration.The plant that somatic embryo is sprouted as shown in Figure 4.
Because somatic embryo has dipolar characteristics, so ripe cotyledonary embryos can form cotyledon and root, becomes complete seedling simultaneously, then well solved in the life of somatic embryos of coniferous trees fetal hair the difficult problem of taking root.
Embodiment 16
The sprouting stage of somatic embryo: the somatic embryo that embodiment 14 is obtained is sprouted, use the 1/4LM minimal medium, drying time is set to be respectively 0,1,4,7 and 10 day, do not add any hormone in medium, and fixing additional 2% sucrose, other medium components and condition of culture are sprouted and be the results are shown in following table after 15, three weeks with embodiment:
| Drying time (my god) | 0 | 1 | 4 | 7 | 10 |
| Germination rate (%) | 16 | 19 | 60 | 33 | 36 |
The essence of drying and other treatment, be the somatic embryo that makes to reach morphological maturity, experiences a suitable dry environment, in certain limit, by losing moisture, to realize somatic embryo, enters physiological ripening, thereby be conducive to the process that somatic embryo is sprouted.As seen from the above table, along with the prolongation of drying time, the germination rate of somatic embryo presents the trend that first raises and reduce afterwards, when drying and other treatment in the time of 4 days, can make the germination rate of somatic embryo bring up to 60% by 16%, this is a very large breakthrough concerning the sprouting of conifer somatic.
Embodiment 17
The sprouting stage of somatic embryo: the somatic embryo that embodiment 13 is obtained is sprouted, use the 1/4LM minimal medium, PEG4000 concentration is set and is respectively 0,2.5%, 5%, 7.5%, 10% and 15%, do not add any hormone in medium, and fixing additional 2% sucrose, other medium components and condition of culture are sprouted and be the results are shown in following table after 15, three weeks with embodiment:
| PEG4000 concentration (%) | 0 | 2.5 | 5 | 7.5 | 10 | 15 |
| Germination rate (%) | 23 | 14 | 16 | 9 | 14 | 6 |
As seen from the above table, after adding PEG4000, the germination rate of somatic embryo all has reduction in various degree, in this enforcement, not add PEG4000, can obtain higher somatic embryo germination rate.
In the present invention, the stages occurred for somatic embryo has the combination of clear and definite medium scheme, hormone and other nutriments.The inductivity of thick branch dragon spruce embryo callus on average reaches more than 60%, reaches as high as more than 95%; The average frequency of embryonic callus induction of the Picea seeds of this result and existing bibliographical information 30% left and right is compared, and has obtained great raising.The somatic differentiation frequency of thick branch dragon spruce, the inductivity of the mature somatic embryo under single factor effect surpasses 100/gram, and the inductivity of the mature somatic embryo under the effect of best of breed factor reaches 583/gram; The existing the highest approximately report of 40/gram of document has been compared great raising.; The germination rate of mature somatic embryo reaches as high as 60%.Simultaneously, the differentiation of somatic embryo presents high synchronized phenomenon, and the somatic embryo of differentiation is almost all in same cotyledonary embryos period, and this is very crucial for thick branch dragon spruce body embryo generation technique application production practices.
Adopt technology provided by the invention, for the batch production asexual multiplication seedling of thick branch dragon spruce provides a kind of method that cycle is short, reproduction rate is high, with low cost; Broken through the natural ripening rate of thick branch dragon spruce low, the cottage propagation difficulty, the sapling multiplication technology can't meet the restriction of clonal planting needs.
Embodiment 18
The thick branch dragon spruce immature zygotic embryos that mid-June to late June gathers of take is explant, and the embryonic callus induction medium is 1/2LM+10 μ M2, and 4-D+5 μ M6-BA adds 1gL
-1the natural complex casein hydrolysis, 1% sucrose, 500mgL
-1glutamine and 0.2% plant gel.Dark condition is cultivated after 7 weeks (other conditions are with embodiment 2), and frequency of embryonic callus induction reaches more than 60%.
Subsequently, embryo callus is placed on semisolid culturemedium and is bred, semi-solid proliferated culture medium is 1/2LM+10 μ M2, and 4-D+5 μ M6-BA adds 1gL
-1the natural complex casein hydrolysis, 1% sucrose, 500mgL
-1glutamine and 0.2% plant gel.Condition of culture is all identical with induction period.Place the embryo callus of 7 diameter 0.5cm sizes in each culture dish (diameter 90mm), switching in two weeks once, during switching, the embryo callus amount of growth can double, and within 2 months, can set up the semi-solid propagation of the embryo callus system of certain scale later.
With the embryo callus obtained, breeding is a minute formed material, and differential medium is 1/2LM+100 μ M ABA+5%PEG4000+3% sucrose+0.1% active carbon+0.4% gel, and additional concentration is 1gL
-1caseinhydrolysate, 500mgL
-1glutamine, add before gel pH be adjusted to 5.8, sterilizing 20 minutes under 121 ℃, pressure 101kp subsequently.Differentiation phase, tiling one deck Whatman filter paper in differential medium, under aseptic condition, tile the embryo callus of about 1g left and right on filter paper, notices that tiling evenly.24 ± 1 ℃ of lower dark culturing.After 6～7 weeks, the quantity that differentiates high synchronized somatic embryo reach 500/more than gram.
The mature somatic embryo of acquisition is sprouted to cultivation, and germination medium is 1/4LM+2% sucrose+0.4% gel, does not add any hormone in medium, also without PEG4000, participates in.Additional 1gL
-1caseinhydrolysate, 500mgL
-1glutamine.Add before gel pH is adjusted to 5.8, sterilizing 20 minutes under 121 ℃, pressure 101kp subsequently.Before sprouting, by somatic embryo process mummification pretreatment 4 days, make germination rate bring up to 60%, reach international similar research level.In addition, in the germination process of somatic embryo, somatic embryo is placed horizontally to the semisolid culturemedium surface, culture dish inclination 45° angle is placed, and is conducive to cotyledon stretching, extension, plumular axis and radicle elongation; Simultaneously, the radicle of growth is attached to media surface, and while having avoided transplanting, the tender shoot root of children is impaired, is conducive to transplant survival.
The plant somatocyte embryo generation technique is as a kind of vegetative propagation technique, whole technical system mainly comprises the inducing and 5 parts such as the differentiation of propagation, mature somatic embryo and sprouting of selection, embryo callus of explant, and the exploration in these stages itself is had to certain process; Therefore, method commonly used is also substantially identical.But the difference maximum is also the most key, the effect that the varying level of each influence factor and the combination of each factor produce varies; Simultaneously, the standardization of experimental implementation, the validity of each factor level design and high efficiency and the reliability of result are also widely different, and therefore, almost each seeds has a whole set of exclusive somatic embryo generation medium.In the application's experimental study, because thick branch dragon spruce not yet has the report of body embryo aspect research, with reference on the basis of a large amount of similar researchs, experimental design is in line with making every effort to the principle of to greatest extent each factor that affects the somatic embryo generation being inquired into, simultaneously according to the result of a large amount of preliminary experiments, the experiment water of each factor has been carried out to reasonable setting.Make result of study stable, operability and repeatability are stronger.
In the domestic patent of having reported at present, aspect research strategy, only have the comparatively similar of balfour spruce, because be the cause that same seminar conducts a research, however, the difference as a result that different phase is processed is also very large.In disclosed domestic literature, there is " explant sterilization " loaded down with trivial details, inefficient problem; Differentiation is common to be adopted " block differentiation ", and this differentiation mode is large to embryo callus materials demand amount, and the quantity of the somatic embryo of differentiation generation is few, and the synchronization degree is low; In the sprouting stage, how just domestic literature text description, to concrete Data Source and the rare report of analysis.In addition, somatic embryo occurs to expand outside numerous technology as a Plants, what in research process, obtain enriches excellent material, the mechanism research that somatic embryo is grown provides desirable material, domestic very few about mechanism research, the regenerating system of truly not realizing the seeds of reporting is described, it is not mature enough, stable that body embryo method seems.Therefore, the domestic existing bibliographical information about the Picea seeds, reference value is limited.The present inventor has adopted the LM medium as minimal medium, adopt the technology such as filter paper differentiation rather than the block differentiation of domestic callus commonly used in the embryo differentiate process, make result of study compare with domestic and international existing report, realized progressive significantly.
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 improvement to it, this will be apparent to those skilled in the art.Therefore, without departing from theon the basis of the spirit of the present invention, these modifications or improvements, all belong to the scope of protection of present invention.
Claims (10)
1. a thick branch dragon spruce somatic embryo occurs and plant regeneration method, comprising: gather the clonal cone of thick branch dragon spruce open pollination, 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.
2. method according to claim 1, is characterized in that, the described embryonic callus induction stage: adopt the 1/2LM medium, additional 2,4-Benzene Chloride fluoroacetic acid, 5～15 μ M, 6-benzylaminopurine 2.5～10 μ M; Described 2,4-Benzene Chloride fluoroacetic acid is 10 μ M preferably, and the 6-benzylaminopurine is 5 μ M preferably.
3. method according to claim 2, is characterized in that, in embryonic callus induction stage 1/2LM medium used, also comprises: the sucrose that concentration is 1%, 500mgL
-1glutamine, 1gL
-1casein hydrolysis and 0.2% plant gel.
4. according to the described method of claim 1～3 any one, it is characterized in that, the described embryo callus multiplicative stage: adopt the 1/2LM medium, 2 of additional 3.75～10 μ M, the 6-benzylaminopurine of 4-Benzene Chloride fluoroacetic acid and 1.25～7.5 μ M, preferably 2 of 3.75～5 μ M, the 6-benzylaminopurine of 4-Benzene Chloride fluoroacetic acid and 1.25～3.75 μ M, more preferably 2 of 5 μ M, the 6-benzylaminopurine of 4-Benzene Chloride fluoroacetic acid and 3.75 μ M.
5. method according to claim 4, is characterized in that, the 1/2LM medium that the embryo callus multiplicative stage is used, and cell volume and culture fluid volume ratio are 1:3～1:15, preferably 1:5～1:15, more preferably 1:7.
6. according to the described method of claim 4 or 5, it is characterized in that, in embryo callus multiplicative stage 1/2LM medium used, the sucrose concentration of interpolation is 0.5～3.0%, preferably 1～1.5%, more preferably 1%;
Also comprise: 500mgL
-1glutamine, 1gL
-1casein hydrolysis.
7. according to the described method of claim 1～6 any one, it is characterized in that the described somatic embryo stage of ripeness: adopt the 1/2LM medium, the abscisic acid of additional 31～123 μ M, preferably 62～123 μ M, more preferably 92～123 μ M, most preferably 100～108 μ M.
8. method according to claim 7, is characterized in that, in the somatic embryo stage of ripeness middle 1/2LM medium used, also comprises: Macrogol 4000, and concentration is 0～7.5%, preferably 3.5～7.5%, more preferably 5～7.5%, most preferably 5%;
Add 1～9% sucrose, preferably 1～4.5%, more preferably 1～3%, most preferably 3%;
Add 0～0.5% active carbon, preferably 0.1～0.5%, more preferably 0.1%;
Add 0.2～0.8% plant gel, preferably 0.2～0.6%, more preferably 0.4%;
Also comprise: 500mgL
-1glutamine and 1gL
-1caseinhydrolysate.
9. according to the described method of claim 1～8 any one, it is characterized in that, described somatic embryo is sprouted the stage: adopt the 1/4LM medium, the concentration of activated carbon of interpolation is 0～0.5%, preferably 0.1～0.4%, more preferably 0.2%;
Add 0～15% Macrogol 4000, preferably 0～10%, more preferably 0%;
Add 1～15% sucrose, preferably 1～7%, more preferably 1～4%, most preferably 2%;
Also comprise: 500mgL
-1glutamine, 1gL
-1caseinhydrolysate and 0.4% plant gel.
10. method according to claim 9, is characterized in that, somatic embryo is sprouted stage 1/4LM medium used, and drying time is 0～21 day, preferably 1～10 day, and more preferably 4～10 days, most preferably 4 days.
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| CN110999785B (en) * | 2019-11-28 | 2021-08-31 | 内蒙古蒙荣园林绿化工程有限责任公司 | Synthetic method of artificial seeds of picea asperata |
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