CN103461119B - PIECA ASPERATA somatic embryo occurs and plant regeneration method - Google Patents
PIECA ASPERATA somatic embryo occurs and plant regeneration method Download PDFInfo
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- CN103461119B CN103461119B CN201310364926.4A CN201310364926A CN103461119B CN 103461119 B CN103461119 B CN 103461119B CN 201310364926 A CN201310364926 A CN 201310364926A CN 103461119 B CN103461119 B CN 103461119B
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Abstract
The present invention relates to a kind of PIECA ASPERATA somatic embryo to occur and plant regeneration method, comprising: gather PIECA ASPERATA open pollination clonal cone, isolate immature zygotic embryos and carry out body embryo and cultivate; Described body embryo generation cultivation comprises four-stage: embryonic callus induction stage, embryo callus multiplicative stage, somatic embryo maturation stage and somatic embryo sprout the stage.The present invention belongs to together in the world with existing or the research report of similar seeds is compared, frequency of embryonic callus induction greatly improves, differentiation capability and the sprouting ability of somatic embryo also greatly improve, screening simultaneously obtains keeping for a long time and enrichment procedure of embryo callus, greatly reduces the risk losing embryo.
Description
Technical field
The present invention relates to cell engineering sapling multiplication field in forestry, particularly, relate to a kind of PIECA ASPERATA somatic embryo and occur and plant regeneration method.
Background technology
PIECA ASPERATA (Picea asperata Mast) is Pinaceae, Picea seeds, is domesticly commonly called as dragon spruce, also cries large fruit dragon spruce, is 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.Bark ficelle, is cleaved into irregular scale or slightly thick fast sheet comes off; Sprig has the pubescence dredging raw or close life, or without hair, light brown yellow, isabelline or reddish tan time annual, and pulvinus has white powder, or white powder is not obvious; Hibernaculum is conical, and have resin, base portion expands.The bar shaped of needle four prismatic, long 1 ~ 2cm, wide 1 ~ 1.5mm, microbend, the micro-point of tip or anxious point, cross section four prismatic.The cylindric square of cone is circular or cylindrical, and upper end is gradually narrow, green before ripe, filbert or Chestnut time ripe, long 5 ~ 16cm, footpath 2.5 ~ 3.5cm; Middle part fruit scale obovate; Seed falls oval, and be about 4mm, fructus forsythiae is about 1.5cm, and seed wing is filbert, and the shape of falling ovum square is circular; Cotyledon 6 ~ 7 pieces.In 4 ~ May of florescence, cone is ripe for 9 ~ October.For China endemic species, be mainly distributed in the Subalpine region area in west Sichuan 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.PIECA ASPERATA system shallow root seeds, slightly resistance to the moon, the environment that ability is dry and cold.Timber yellow-white, more light and soft, texture is straight, and structure is thin, flexible.Papermaking, building, furniture and the xylem fibre raw material of industry etc. can be made and use material.Material is excellent, and growth is fast, and strong adaptability is the Major Tree Species Planted of area.At present, artificial afforestration seedling is mainly through Seedling propagation, but PIECA ASPERATA solid evening, seed maturity rate is low, and with serious " biennial bearing " phenomenon, supply falls short of demand to cause PIECA ASPERATA good seed.
Plant somatocyte embryo generation technique, refers in vitro, and the somatic cell broken up under certain environmental conditions, develops into the process of the embryoid similar with zygotic embryo.Plant somatocyte embryo utilizes one of cell engineering means important channel realizing plant regeneration, utilize artificial means can realize plant to greatest extent to expand numerous measure as a kind of, especially for being difficult to cottage propagation and breeding cycle longer coniferous tree seeds, there is huge using value.Meanwhile, 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 disclosing the genesis mechanism research of plant embryos and carrying out Study on Genetic Transformation and provide a kind of model study system.
Coniferous species is distribution one of class seeds the most widely in world wide, owing to usually having excellent wood property, as the of paramount importance industrial cut stock seeds of the developed countries and regions such as North America and Northern Europe.But because it is different from phanerogamic characteristic, coniferous species is more responsive to isolated condition, be the class seeds that in somatic embryo generation research process, difficulty is maximum.According to incompletely statistics, up to now, the whole world have an appointment more than 150 seeds achieve somatic embryo occur, wherein coniferous species only have an appointment 50 kinds achieve somatic embryo occur and plant regeneration.
At present, occur and plant regeneration method about PIECA ASPERATA somatic embryo, there is no bibliographical information both at home and abroad.Therefore, develop a kind of PIECA ASPERATA somatic embryo efficiently and occur and plant regeneration method, for realizing the factorial seedling growth based theoretical of PIECA ASPERATA somatic embryo and providing particularly necessary practicable method; Meanwhile, also good experimental system can be set up for the biotechnology breeding of PIECA ASPERATA, Germ-plasma resources protection, Fast-propagation, genetic transformation.
Summary of the invention
In order to solve the problems of the prior art, the object of this invention is to provide a kind of PIECA ASPERATA somatic embryo and occurring and plant regeneration method.
A kind of PIECA ASPERATA somatic embryo provided by the invention occurs and plant regeneration method, comprising: gather the clonal cone of PIECA ASPERATA open pollination, isolates immature zygotic embryos (namely carrying out the selection of explant) and carries out body embryo and cultivate; Described body embryo generation cultivation comprises four-stage: embryonic callus induction stage, embryo callus multiplicative stage, somatic embryo maturation stage and somatic embryo sprout the stage.
Wherein, in the selection of described explant, the immature zygotic embryo of growth adopting PIECA ASPERATA open pollination is material, and it is the last ten-days period in mid-June to September that cone gathers the date, preferred the middle ten days and the last ten days in June.Isolate the induction that immature zygotic embryos carries out embryo callus, whole Induction Process aseptically carries out.
Wherein, the described embryonic callus induction stage: adopt 1/2LM minimal medium, additional auximone 2,4-Benzene Chloride fluoroacetic acid (2,4-dichlorophenoxyacetic acid, hereinafter referred to as 2,4-D) concentration be 5 ~ 15 μMs, the concentration of basic element of cell division 6-benzylaminopurine (benzylaminmopurine, hereinafter referred to as 6-BA) is 2.5 ~ 10 μMs.Described 2,4-D preferably 10 μMs, 6-BA preferably 5 μMs.
Further, also comprise in the 1/2LM minimal medium that the embryonic callus induction stage is used: concentration is the sucrose of 1%, 500mgL
-1glutamine, 1gL
-1casein hydrolysis and 0.2% plant gel, the highest embryonic callus induction frequency can be obtained under this condition.
Wherein, the described embryo callus multiplicative stage: adopt 1/2LM liquid nutrient medium, additional hormone is the 6-BA of 3.75 ~ 10 μMs 2,4-D and 1.25 ~ 7.5 μMs, preferably 3.75 ~ 5 μMs 2, the 6-BA of 4-D and 1.25 ~ 3.75 μM, more preferably the 6-BA of 2,4-D and 3.75 μMs of 5 μMs, 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, preferred 1:5 ~ 1:15, more preferably 1:7; Maximum embryo callus harvest yield can be obtained when volume ratio is 1:7.
Further, in the 1/2LM minimal medium (i.e. inducing culture) that the embryonic callus induction stage is used, the concentration of 2,4-D is preferably 2 times of 6-BA; In the 1/2LM liquid nutrient medium (i.e. proliferated culture medium) that the described embryo callus multiplicative stage is used, 2, the concentration of a little higher than 6-BA of concentration of 4-D, and in proliferated culture medium hormone concentration comparatively inducing culture obviously reduce, this kind of hormone combinations is conducive to the maintenance of the callus of height embryo.
Wherein, in the 1/2LM liquid nutrient medium that the described embryo callus multiplicative stage is used, adopt sucrose as carbon source material, concentration is 0.5 ~ 3.0%, preferably 1 ~ 1.5%, most preferably 1%(most preferably time consistent with sucrose concentration in inducing culture).
Wherein, in the 1/2LM liquid nutrient medium (i.e. proliferated culture medium) that the described embryo callus multiplicative stage is used, very crucial is exactly a bit reduce the cell contamination frequency suspended when cultivating to greatest extent.
In the present invention, embryo callus is placed in the conical flask of the 250ml that liquid nutrient medium is housed and cultivates, need conical flask autoclaving before cultivation, with after the sealing of ventilation sealed membrane after inoculation, need the aseptic newspaper of sealing two layers again, greatly can reduce the risk of cell contamination like this, make Contamination rate control within 1%.
Wherein, in the 1/2LM liquid nutrient medium that the described embryo callus multiplicative stage is used, also comprise: 500mgL
-1glutamine, 1gL
-1casein hydrolysis.When carrying out Multiplying culture, shaking speed is 110 revs/min.
The method of the invention, the hormone concentration that the embryonic callus induction stage adopts can obtain high embryonic callus induction frequency.In the Multiplying culture of embryo callus, what the present invention adopted is liquid suspension culture mode, is optimized, greatly can maintains the embryo sexuality of embryo callus to condition of culture, reduces the risk that it loses embryo sexuality; Meanwhile, can obtain in a large number and the embryo callus of high degree of homogenous, be conducive to the differentiation of later stage high level of synchronization somatic embryo.
Wherein, the described somatic embryo maturation stage: adopt 1/2LM minimal medium, additional hormone is abscisic acid (the abscisic acid of 31 ~ 123 μMs, hereinafter referred to as ABA), preferably 62 ~ 123 μMs, more preferably 92 ~ 123 μMs, preferably 100 ~ 108 μMs further; Under the ABA process of 100 μMs, can obtain a large amount of and that synchronization degree is higher mature somatic embryo, such cost effective is conducive to again the sprouting of later stage mature somatic embryo.
Wherein, in the middle 1/2LM minimal medium that the described somatic embryo maturation stage is used, also comprise: osmotic adjustment Macrogol 4000 (be called for short PEG4000), concentration is 0 ~ 7.5%, preferably 3.5 ~ 7.5%, more preferably 5 ~ 7.5%, more preferably 5%.
Wherein, in the middle 1/2LM minimal medium that the described somatic embryo maturation stage is used, adopt the sucrose of 1 ~ 9%, preferably 1 ~ 4.5%, more preferably 1 ~ 3%, further preferably 3%.
Wherein, in the middle 1/2LM minimal medium that the described somatic embryo maturation stage is used, adopt the active carbon of 0 ~ 0.5%, preferably 0.1 ~ 0.5%, more preferably 0.1%.
Wherein, in the middle 1/2LM minimal medium that the described somatic embryo maturation stage is used, adopt the plant gel of 0.2 ~ 0.8%, preferably 0.2 ~ 0.6%, more preferably 0.4%.
Wherein, in the middle 1/2LM minimal medium that the described somatic embryo maturation stage is used, also comprise: 500mgL
-1glutamine and 1gL
-1caseinhydrolysate.
Wherein, the condition of culture in described embryonic callus induction stage, embryo callus multiplicative stage and somatic embryo maturation stage can be all: before adding gel, pH is adjusted to 5.8, subsequently 121 DEG C, pressure 101kp high temperature, sterilizing 20 minutes under condition of high voltage; Cultivation temperature 24 ± 1 DEG C, dark condition is cultivated.
The method of the invention, by testing the many factors affecting somatic embryo inducement and maturation, the best maturation medium combination that screening obtains, be conducive to realizing a large amount of of PIECA ASPERATA somatic embryo and high synchronization generation, be conducive to inducing the somatic embryo maturation produced, the realization for PIECA ASPERATA factorial seedling growth provides the most key guarantee.
Wherein, described somatic embryo sprouts the stage: adopt 1/4LM minimal medium, concentration of activated carbon is 0 ~ 0.5%, and preferably 0.1 ~ 0.4%, more preferably 0.2%.
Wherein, the 1/4LM minimal medium that the described somatic embryo sprouting stage is used, adopts drying time to be 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 the PEG4000 of 0 ~ 15%, and preferably 0 ~ 10%, more preferably 0%, namely 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 the sucrose of 1 ~ 15%, 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 at Semi-solid cell culture primary surface, culture dish inclination 45° angle is placed, and is conducive to cotyledon stretching, extension, plumular axis and radicle and extends; Meanwhile, the radicle of growth is attached to media surface, and when avoiding 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 DEG C; First week (with 7 days for the cycle), dark culturing; Second week (with 7 days for the cycle), 20 μm of olm
-2s
-1under illumination, and hide with one deck paper (newspaper), the photoperiod is that 16h illumination and 8h are dark; Subsequently, 50 μm of olm
-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 glutamine and the ABA non-refractory of use, adopt the mode of filtration sterilization, other all the components all adopt the sterilizing of HTHP mode.
Method of the present invention, before carrying out embryonic callus induction to immature zygotic embryos, also comprises: carry out disinfection to prematurity cone, then under aseptic environment, takes out immature zygotic embryos.
Wherein, described disinfection way is: sterilize 15 ~ 20min to PIECA ASPERATA prematurity cone (i.e. the clonal cone of above-mentioned PIECA ASPERATA open pollination) to utilize absolute ethyl alcohol, then with aseptic water washing at least 2 times.This kind of disinfection way can make Contamination rate control within 0.1%, can meet the needs of practical operation completely, and simple to operate, efficient, cost is low, environmental pollution is little.
The percent concentration " % " occurred in the present invention all refers to the grams needing the material added in quality-volumetric concentration g/100mL(and 100mL solution).
Up to now, the external tender stem apex of children that adopts is that explant carries out inducing and can only induce embryo callus at most, all can not be divided into somatic embryo.Present inventor's cotyledon, plumular axis and radicle attempted with seed sprouting is that explant is induced, and can induce callus, but frequency of embryonic callus induction is extremely low, and can not continues normal proliferative, can not carry out the differentiation of somatic embryo.So the present invention utilizes whole zygotic embryo as explant, carry out induction contrast to the explant of development in different stages, screening obtains optimum immature zygotic embryos developmental stage, meets the needs of research and actual production.
In addition, the application is by a large amount of experiments, and the specified conditions with plant regeneration occur the somatic embryo filtered out for PIECA ASPERATA, are conducive to the induction frequency improving embryo callus; Be conducive to the embryo sexuality maintaining embryo callus, reduce the risk losing embryo sexuality; Be conducive to realizing a large amount of of somatic embryo and the synchronized acquisition of height; Be conducive to the germination efficiency improving somatic embryo.
Method provided by the invention, special low and there is serious " biennial bearing " phenomenon for the natural ripening rate of PIECA ASPERATA, cuttage root-taking difficulty, sapling multiplication technology cannot meet the present situation of large area afforestation needs, and the technology with plant regeneration occurs the somatic embryo finding a kind of PIECA ASPERATA choiceness.Belong to together in the world with existing or similar seeds research report compare, frequency of embryonic callus induction greatly improves, differentiation capability and the sprouting ability of somatic embryo also greatly improve, screening simultaneously obtains keeping for a long time and enrichment procedure of embryo callus, greatly reduces the risk losing embryo.Research maintains the leading position, for realizing the factorial seedling growth based theoretical of PIECA ASPERATA somatic embryo and providing particularly necessary practicable method; Meanwhile, also good experimental system can be set up for the biotechnology breeding of PIECA ASPERATA, Germ-plasma resources protection, Fast-propagation, genetic transformation.
Accompanying drawing explanation
Fig. 1 is the immature zygotic embryos of PIECA ASPERATA.
Fig. 2 is the embryo callus of PIECA ASPERATA.
Fig. 3 is the mature somatic embryo of PIECA ASPERATA.
Fig. 4 is the plant that the somatic embryo of PIECA ASPERATA is sprouted.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
The present invention plant gel gellan used gum, adopts Sigma brand.It is a kind of epoxy resin, and the effect with agar is identical, 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, same employing Sigma brand, compare with the active carbon that open market is sold, 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 buied 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
2o(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, macroelement, trace element and molysite are reduced by half, other are constant.The 1/4LM minimal medium that the present invention occurs is exactly that macroelement, trace element and molysite are reduced to 1/4, and other are constant to above-mentioned LM minimal medium.The 1/2LM liquid nutrient medium that the present invention occurs is exactly that macroelement, trace element and molysite are reduced by half, other are constant to above-mentioned LM minimal medium, does not add gel in medium simultaneously.The 1/2LM semisolid culturemedium that the present invention occurs is exactly that macroelement, trace element and molysite are reduced by half, other are constant to above-mentioned LM minimal medium, adds gel in medium simultaneously.
Wherein, first prepare mother liquor during actual use, then dilute use.Macroelement is become 100 times with the mother liquor of molysite, and trace element is mixed with 1000 times, and inositol and organic salt are mixed with 500 times.CaCl in macroelement
22H
2o and other compositions combine and form precipitation, it are prepared separately.
Content of the present invention is the comprehensive of test data between 2011 ~ 2013 years, and experimental result is stable, reliable.Described experiment material picks up from the national germplasm resource bank of Gansu Province's Xiao Longshan forestry pilot office forestry research institute dragon spruce.
Embodiment 1
Isolate immature zygotic embryos: gather the prematurity cone that open pollination in about 1 day May produces, start at the beginning of 6 months to carry out the collection of prematurity cone, gathered once every 7 ~ 10 days, gather 15 times altogether, 12 genotype, place the cone low temperature under 4 DEG C of conditions under adopting.With absolute ethyl alcohol process cone 15min, aseptic water washing 2 times.Under aseptic condition, take out seed and peel off seed coat and endosperm, immature zygotic embryos (as shown in Figure 1) being carried out the induction of embryo callus.
Embodiment 2
In the embryonic callus induction stage: immature zygotic embryos embodiment 1 obtained carries out embryonic callus induction, adopt 1/2LM minimal medium, additional hormone concentration is as shown in the table respectively:
| Process number | 2,4-D/μM | 6-BA/μM |
| 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 |
Further, 1gL is added
-1natural complex casein hydrolysis, in medium containing 1% sucrose, 500mgL
-1glutamine and 0.2% plant gel (gellan gum, Sigma).Add before gel and pH be adjusted to 5.8, subsequently 121 DEG C, pressure 101kp high temperature, sterilizing 20 minutes under condition of high voltage.About 35ml semisolid culturemedium is filled in the glass culture dish of diameter 90mm.Cultivation temperature 24 ± 1 DEG C, dark condition is cultivated, to inducing embryo callus.
Immature zygotic embryos is inoculated in callus inducing medium after 2-3 days, and zygotic embryo starts to start, and be embodied in embryo and expand, plumular axis base portion has 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 grown up gradually together with non embryogenic callus subsequently, embryo callus separately can be bred separately after 6-7 week.Non embryogenic callus is many to be differentiated by plumular axis, and fast, majority is brown particles shape, and differentiation phase can not produce normal somatic embryo in growth; Embryo callus majority " spot-like projections " of being broken up by cotyledon is transformed, and is embodied in white, transparent, thickness, thread, and possesses the ability differentiating normotrophic somatic embryo.
Can obtain the highest embryonic callus induction frequency with the immature zygotic embryos of mid-June to late June, average Induce of embryoid rate is more than 60%, and high Induce of embryoid genotype can reach more than 95%.Now, zygotic embryo is grown and is in the early stage cotyledonary embryos stage.With (as following embodiment 3) under the 6-BA process of 10 μMs 2,4-D and 5 μMs, the frequency of embryonic callus induction of acquisition is the highest, and the combination of taking second place is 2,4-D15 μM and 6-BA5 μM, and the poorest combination is 2,4-D5 μM and 6-BA10 μM; The too high or too low induction being all unfavorable for embryo callus of hormone concentration.
Embodiment 3
In the embryonic callus induction stage: immature zygotic embryos embodiment 1 obtained carries out embryonic callus induction, adopt 1/2LM minimal medium, additional hormone concentration is respectively 2,4-D10 μM and 6-BA5 μM, adds 1gL
-1natural complex casein hydrolysis, in medium containing 1% sucrose, 500mgL
-1glutamine and 0.2% plant gel.Add before gel and pH be adjusted to 5.8, subsequently 121 DEG C, pressure 101kp high temperature, sterilizing 20 minutes under condition of high voltage, cultivation temperature 24 ± 1 DEG C, dark condition is cultivated, to inducing embryo callus.Under the collection date of the best, multiple genotypic average inductivity reaches more than 64%.
Embodiment 4
The multiplicative stage of embryo callus: bred by the embryo callus that embodiment 3 obtains, use 1/2LM semisolid culturemedium, growth hormone 2,4-D concentration is 10 μMs, and basic element of cell division 6-BA concentration is 5 μMs, and 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 pieces of diameter 0.5cm sizes in each culture dish (diameter 90mm), once, during switching, embryo callus amount of growth can double many, and namely monolithic diameter about 1 ~ 1.5cm(as shown in Figure 2 in switching in two weeks).
Embodiment 5
The multiplicative stage of embryo callus: the embryo callus that embodiment 4 obtains is got about 1g.Be placed in 250ml conical flask, in bottle, place 50ml liquid nutrient medium, carry out liquid suspension propagation.1/2LM minimal medium, does not add gel.2,4-D concentration is 10 μMs, and 6-BA concentration is 5 μMs, and sucrose concentration is 1%.Casein hydrolysis concentration is 1gL
-1, glutamine is 500mgL
-1, shaking speed is 110 revs/min, cultivation temperature 24 ± 1 DEG C, and dark condition is cultivated.After 10 days, embryo callus is transferred, the free callus shaken up of just transferring during switching, switching medium is identical; So, the suspension cell line of embryo callus is namely obtained after 1 month.
Embryo callus is placed in the conical flask of the 250ml that liquid nutrient medium is housed and cultivates, need conical flask autoclaving before cultivation, with after the sealing of ventilation sealed membrane after inoculation, need the aseptic newspaper of sealing two layers again, greatly can reduce the risk of cell contamination like this, make Contamination rate control within 1%.
Embodiment 6
The multiplicative stage of embryo callus: be material with the embryo callus suspension cell obtained in embodiment 5, cell volume (leave standstill 1h) is set and is respectively 1:5,1:7,1:9,1:12 and 1:15 with the ratio (inoculum concentration) of culture fluid.Be placed in 250ml conical flask by 50ml liquid nutrient medium and cultivate, medium component and condition of culture are with embodiment 5.Continue cultivation 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, suspension propagation, after 9 days, can obtain the fresh weight of 24.696g/100ml, adds 4.24 times when more just inoculating.
Embodiment 7
The multiplicative stage of embryo callus: with in embodiment 5 obtain embryo callus suspension cell be material, arranging cell volume (leaving standstill 1h) is 1:7 with the ratio of culture fluid, reset growth hormone 2, the concentration of 4-D is respectively 3.75,5,7.5 and 10 μMs, and 6-BA concentration is fixed as 5 μMs.Be placed in 250ml conical flask by 70ml liquid nutrient medium and cultivate, other medium components and condition of culture are with embodiment 5.Continue cultivation after 9 days, the amount of growth of embryo callus is as shown in the table:
| 2,4-D concentration (μM) | 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 the raising of growth hormone 2,4-D concentration, the growth of embryo callus presents the trend that existing increase reduces afterwards.When 2,4-D concentration is 5 μMs, can obtain maximum embryo callus amount of growth, suspension propagation, after 9 days, can obtain the fresh weight of 24.034g/100ml, adds 4.54 times when more just inoculating.
Embodiment 8
The multiplicative stage of embryo callus: with in embodiment 5 obtain embryo callus suspension cell be material, arranging cell volume (leaving standstill 1h) is 1:7 with the ratio of culture fluid, the concentration resetting basic element of cell division 6-BA is respectively 1.25,2.5,3.75,5 and 7.5 μMs, 2,4-D concentration is fixed as 5 μMs.Be placed in 250ml conical flask by 70ml liquid nutrient medium and cultivate, other medium components and condition of culture are with embodiment 5.Continue cultivation after 9 days, the amount of growth of embryo callus is as shown in the table:
| 6-BA concentration (μM) | 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 μMs, can obtain maximum embryo callus amount of growth, suspension propagation, after 9 days, can obtain the fresh weight of 13.571g/100ml, adds 4.06 times when more just inoculating.Further, in the multiplicative stage of embryo callus, additional hormone is the 6-BA of 5 μMs 2,4-D and 3.75 μMs, and effect is optimum, is the best hormone combinations that can obtain embryo callus propagation.
Embodiment 9
The multiplicative stage of embryo callus: be material with the embryo callus suspension cell obtained in embodiment 5, cell volume (leave standstill 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 μMs, and 6-BA concentration is 5 μMs.Be placed in 250ml conical flask by 70ml liquid nutrient medium and cultivate, other medium components and condition of culture are with embodiment 5.Continue cultivation 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, suspension propagation, after 9 days, can obtain the fresh weight of 15.566g/100ml, adds 4.44 times when more just inoculating.
Note: the embryo callus suspension cell of the initial inoculation that embodiment 6 ~ 9 is used is material, and respective fresh weight is different, is as the criterion with the actual fresh weight used of each embodiment.
Embodiment 10
In the stage of ripeness of somatic embryo: embryo callus embodiment 4 obtained carries out the maturation culture of somatic embryo, adopt 1/2LM minimal medium.ABA concentration is set and is respectively 31,46,62 and 92 μMs, 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 induction and multiplicative stage, and concentration is 1gL
-1, the concentration of glutamine is 500mgL
-1, add before gel and pH be adjusted to 5.8, subsequently 121 DEG C, sterilizing 20 minutes under pressure 101kp.Differentiation phase, tile in differential medium one deck Whatman filter paper, under aseptic condition, tiles on filter paper by the embryo callus of about about 1g, notices that tiling evenly.Dark culturing at 24 ± 1 DEG C.
Embryo callus is seeded in differential medium after 2 days, and globular embryo starts to occur, globular embryo increasing number subsequently; After 7 days, Part-spherical embryo changes torpedo-shape embryo into, and torpedo-shape embryo quantity continues to increase subsequently; After 3 weeks, Part-spherical embryo starts to enter cotyledonary embryos state, and after 5 weeks, cotyledonary embryos is obvious, enters maturity state successively.Ripe somatic embryo is healthy and strong, and plumular axis is obvious, and whole somatic embryo is faint yellow.The mature somatic embryo situation obtained under the process of each factor variable concentrations sees the following form:
| ABA concentration (μM) | 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, thus carries out the setting of each condition in the stage of ripeness of somatic embryo.In the present embodiment, best realignment and regrouping is ABA92 μM, PEG40005%, sucrose 3%, active carbon 0.1% and gel 0.4%.
Embodiment 11
The stage of ripeness of somatic embryo: embryo callus embodiment 4 obtained carries out the maturation culture of somatic embryo, adopt 1/2LM minimal medium, ABA concentration is set and is respectively 62,77,85,92,100,108 and 123 μMs, 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 (μM) | 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 μMs, the mature somatic embryo quantity of every gram of embryo callus acquisition is maximum.The somatic embryo synchronization degree obtained under 100 μMs and 108 μMs of ABA conditions is all very high, and variance analysis shows that the quantity variance of the two somatic embryo obtained is not remarkable.Consider the simplicity (100 μMs are easily converted and preparation) of Financial cost and practical operation in addition, finally determine that 100 μMs of ABA are the optium concentration of 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: embryo callus embodiment 4 obtained carries out the maturation culture of somatic embryo, adopt 1/2LM minimal medium, PEG4000 concentration is set and is respectively 4.5%, 5%, 5.5%, 6.5% and 7.5%, fixing additional ABA92 μM, 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 of every gram of embryo callus acquisition is maximum.
It should be noted that embryo callus is before differentiation, not needing on the medium not adding hormone, carry out preculture can normally break up, and can shorten cultivation cycle on the basis used manpower and material resources sparingly.
Embodiment 13
In the stage of ripeness of somatic embryo: embryo callus embodiment 4 obtained carries out the maturation culture of somatic embryo, adopt 1/2LM minimal medium, adopt ABA100 μM, 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), mature somatic embryo quantity (individual/gram) be 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, namely 1/2LM minimal medium is adopted, the ratio arranging cell volume and culture fluid is 1:7, additional 5 μM 2,4-D, the sucrose of 3.75 μMs of 6-BA and 1%, other medium components and condition of culture are with embodiment 5.Cultivate after 9 days, embryo callus is carried out the maturation culture of somatic embryo.Somatic embryo maturation medium component and condition of culture are with embodiment 11.This kind of mode due to embryo callus developmental condition consistent, quality is homogeneous; Not only reduce embryo callus and lose the risk of embryo, can obtain quantitatively and somatic embryo all preferably qualitatively simultaneously, meet the sprouting process of later stage mature somatic embryo.
Embodiment 15
In the sprouting stage of somatic embryo: the somatic embryo that embodiment 13 obtains is sprouted, use 1/4LM minimal medium, sucrose concentration is set and is respectively 1%, 2%, 4%, 7%, 10% and 15%, in medium, do not add any hormone, and additional glutamine 500mgL
-1, 1gL
-1caseinhydrolysate, the plant gel of active carbon 0.1% and 0.4%.Cultivation temperature is 24 ± 1 DEG C.First week, dark culturing; Second week, 20 μm of olm
-2s
-1under illumination, and hide with one deck newspaper, the photoperiod is that 16h illumination and 8h are dark; Subsequently, 50 μm of olm
-2s
-1under illumination, the photoperiod is 16h illumination and 8h dark culturing.
In the germination process of somatic embryo, somatic embryo is placed horizontally at Semi-solid cell culture primary surface, culture dish inclination 45° angle is placed, and is conducive to cotyledon stretching, extension, plumular axis and radicle and extends; Meanwhile, the radicle of growth is attached to media surface, and when avoiding transplanting, the tender shoot root of children is impaired, is conducive to transplant survival.
Sprout after three weeks and the results are shown in following table:
| 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 being all unfavorable for somatic embryo of sucrose concentration.The plant that somatic embryo is sprouted as shown in Figure 4.
Because somatic embryo has dipolar feature, so ripe cotyledonary embryos can form cotyledon and root, becomes complete seedling simultaneously, then well solve in the life of somatic embryos of coniferous trees fetal hair difficult problem of taking root.
Embodiment 16
The sprouting stage of somatic embryo: the somatic embryo that embodiment 14 obtains is sprouted, use 1/4LM minimal medium, drying time is set and is respectively 0,1,4,7 and 10 day, any hormone is not added in medium, and the fixing sucrose of additional 2%, other medium components and condition of culture, with embodiment 15, are sprouted after three weeks and be the results are shown in following table:
| Drying time (my god) | 0 | 1 | 4 | 7 | 10 |
| Germination rate (%) | 16 | 19 | 60 | 33 | 36 |
The essence of drying and other treatment is the somatic embryo making to reach morphological maturity, experiences a suitable dry environment, by losing moisture in certain limit, enters physiological ripening to realize somatic embryo, thus is conducive to the process that somatic embryo sprouts.As seen from the above table, along with the prolongation of drying time, the germination rate of somatic embryo presents the trend first raising and reduce afterwards, when drying and other treatment 4 days, the germination rate of somatic embryo can be made to bring up to 60% by 16%, and this is concerning the sprouting of conifer somatic, is a very large breakthrough.
Embodiment 17
The sprouting stage of somatic embryo: the somatic embryo that embodiment 13 obtains is sprouted, use 1/4LM minimal medium, PEG4000 concentration is set and is respectively 0,2.5%, 5%, 7.5%, 10% and 15%, any hormone is not added in medium, and the fixing sucrose of additional 2%, other medium components and condition of culture, with embodiment 15, are sprouted after three weeks and be the results are shown in following table:
| 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, can obtain higher somatic embryo germination rate not add PEG4000.
In the present invention, there is the combination of clear and definite medium scheme, hormone and other nutriments in each stage occurred for somatic embryo.The inductivity of PIECA ASPERATA embryo callus on average reaches more than 60%, reaches as high as more than 95%; This result is compared with the average frequency of embryonic callus induction about 30% of the Picea seeds of existing bibliographical information, achieves great raising.The somatic differentiation frequency of PIECA ASPERATA, the inductivity of the mature somatic embryo under single factor effect is more than 100/gram, and the inductivity of the mature somatic embryo under the effect of best of breed factor reaches 583/gram; The report that more existing document is the highest about 40/gram has compared great raising.; The germination rate of mature somatic embryo reaches as high as 60%.Meanwhile, 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 PIECA ASPERATA body embryo generation technique application production practices.
Adopt technology provided by the invention, the batch production asexual multiplication seedling for PIECA ASPERATA provides a kind of method that cycle is short, reproduction rate is high, with low cost; Breach the natural ripening rate of PIECA ASPERATA low, cottage propagation difficulty, sapling multiplication technology cannot meet the restriction of clonal planting needs.
Embodiment 18
The PIECA ASPERATA immature zygotic embryos gathered with mid-June to late June is explant, and embryonic callus induction medium is 1/2LM+10 μM of 2,4-D+5 μMs of 6-BA, adds 1gL
-1natural complex casein hydrolysis, the sucrose of 1%, 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, be placed on by embryo callus on semisolid culturemedium and breed, semi-solid proliferated culture medium is 1/2LM+10 μM of 2,4-D+5 μMs of 6-BA, adds 1gL
-1natural complex casein hydrolysis, the sucrose of 1%, 500mgL
-1glutamine and 0.2% plant gel.Condition of culture is all identical with induction period.The embryo callus of 7 pieces of diameter 0.5cm sizes is placed in each culture dish (diameter 90mm), switching in two weeks once, during switching, embryo callus amount of growth can double, and within 2 months, can set up the embryo callus semisolid propagation system of certain scale later.
Breeding with the embryo callus obtained is a point formed material, and differential medium is 1/2LM+100 μM of ABA+5%PEG4000+3% sucrose+0.1% active carbon+0.4% gel, and additional concentration is 1gL
-1caseinhydrolysate, 500mgL
-1glutamine, adds before gel and pH is adjusted to 5.8, subsequently 121 DEG C, sterilizing 20 minutes under pressure 101kp.Differentiation phase, tile in differential medium one deck Whatman filter paper, under aseptic condition, tiles on filter paper by the embryo callus of about about 1g, notices that tiling evenly.Dark culturing at 24 ± 1 DEG C.After 6 ~ 7 weeks, the quantity differentiating high synchronized somatic embryo reaches more than 500/gram.
The mature somatic embryo of acquisition is carried out sprouting to cultivate, germination medium is 1/4LM+2% sucrose+0.4% gel, does not add any hormone in medium, also participates in without PEG4000.Additional 1gL
-1caseinhydrolysate, 500mgL
-1glutamine.Add before gel and pH is adjusted to 5.8, subsequently 121 DEG C, sterilizing 20 minutes under pressure 101kp.Before sprouting by somatic embryo through 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 at Semi-solid cell culture primary surface, culture dish inclination 45° angle is placed, and is conducive to cotyledon stretching, extension, plumular axis and radicle and extends; Meanwhile, the radicle of growth is attached to media surface, and when avoiding transplanting, the tender shoot root of children is impaired, is conducive to transplant survival.
Plant somatocyte embryo generation technique is as a kind of vegetative propagation technique, whole technical system mainly comprises 5 parts such as differentiation and sprouting of the selection of explant, the induction of embryo callus and propagation, mature somatic embryo, has certain process to the exploration in these stages itself; Therefore, conventional method is also substantially identical.But difference is maximum is also the most key, and the effect that the varying level of each influence factor and the combination of each factor produce varies; Meanwhile, the normalization of experimental implementation, high efficiency and the reliability of the validity that each factor level designs and result are also widely different, and therefore, almost each seeds have a whole set of exclusive somatic embryo generation medium.In the experimental study of the application, because PIECA ASPERATA not yet has the report of body embryo aspect research, on basis with reference to a large amount of similar research, experimental design is in line with making every effort to greatest extent on the principle that each factor affecting somatic embryo generation is inquired into, simultaneously according to the result of a large amount of preliminary experiment, reasonable setting is carried out to the experiment water of each factor.Make result of study stablize, operability and repeatability stronger.
In the domestic patent reported at present, in research strategy, only have the comparatively similar of balfour spruce, because be that same seminar conducts a research, however, the result difference of different phase process is also very large.In disclosed domestic literature, there is " explant sterilization " loaded down with trivial details, inefficient problem; Differentiation employing usually " block differentiation ", this differentiation mode is large to embryo callus materials demand amount, and the quantity of the somatic embryo of differentiation generation is few, and synchronization degree is low; In the sprouting stage, the many just text descriptions of domestic literature, to concrete Data Source and the rare report of analysis.In addition, somatic embryo occurs to expand outside numerous technology as a Plants, what obtain in research process enriches excellent material, desirable material is provided to the study mechanism that somatic embryo is grown, domestic very few about study mechanism, illustrate and not truly do not realize the regenerating system that institute reports seeds, body embryo method seems not mature enough, stablizes.Therefore, the domestic existing bibliographical information about Picea seeds, reference value is limited.Present inventor have employed LM medium as minimal medium, the technology such as filter paper differentiation instead of the block differentiation of domestic conventional callus is adopted in embryo differentiate process, result of study is compared with domestic and international existing report, achieves progress 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 amendments to it or improve, 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 (6)
1. PIECA ASPERATA somatic embryo occurs and a plant regeneration method, comprising: gather PIECA ASPERATA open pollination clonal cone, isolates immature zygotic embryos and carries out body embryo and cultivate; Described body embryo generation cultivation comprises four-stage: embryonic callus induction stage, embryo callus multiplicative stage, somatic embryo maturation stage and somatic embryo sprout the stage;
The described embryonic callus induction stage: adopt 1/2LM medium, additional 10 μM of 2,4-Benzene Chloride fluoroacetic acid, 5 μMs of 6-benzylaminopurines;
Also comprise in the 1/2LM medium that the embryonic callus induction stage is used: concentration is the sucrose of 1%, 500mgL
-1glutamine, 1gL
-1casein hydrolysis and 0.2% plant gel;
The described embryo callus multiplicative stage: semi-solid propagation, adopts 1/2LM medium, additional 2, the 4-Benzene Chloride fluoroacetic acid of 10 μMs and the 6-benzylaminopurine of 5 μMs; In the 1/2LM medium that the embryo callus multiplicative stage is used, the sucrose concentration of interpolation is 1%;
Also comprise: 500mgL
-1glutamine, 1gL
-1casein hydrolysis;
The described somatic embryo maturation stage: adopt 1/2LM medium, the abscisic acid of additional 100 μMs;
In the middle 1/2LM medium that the somatic embryo maturation stage is used, also comprise: Macrogol 4000, concentration is 5%;
Add the sucrose of 3%;
Add the active carbon of 0.1%;
Add the plant gel of 0.4%;
Also comprise: 500mgL
-1glutamine and 1gL
-1caseinhydrolysate;
Described somatic embryo sprouts the stage: adopt 1/4LM medium, adds the Macrogol 4000 of 0%;
Add the sucrose of 2%;
Also comprise: 500mgL
-1glutamine, 1gL
-1caseinhydrolysate and 0.4% plant gel;
The 1/2LM medium that the embryo callus multiplicative stage is used, cell volume and culture fluid volume ratio are 1:3 ~ 1:15; Described somatic embryo sprouts the stage: adopt 1/4LM medium, the concentration of activated carbon of interpolation is 0 ~ 0.5%; The 1/4LM medium that the somatic embryo sprouting stage is used, drying time is 4 ~ 10 days.
2. method according to claim 1, 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 1:5 ~ 1:15.
3. method according to claim 2, 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 1:7.
4. method according to claim 1, is characterized in that, described somatic embryo sprouts the stage: adopt 1/4LM medium, the concentration of activated carbon of interpolation is 0.1 ~ 0.4%.
5. method according to claim 4, is characterized in that, described somatic embryo sprouts the stage: adopt 1/4LM medium, the concentration of activated carbon of interpolation is 0.2%.
6. method according to claim 1, is characterized in that, the 1/4LM medium that the somatic embryo sprouting stage is used, and drying time is 4 days.
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| CN110896854B (en) * | 2019-08-22 | 2021-05-11 | 北京林业大学 | Culture method for promoting development and maturation of somatic embryos of Chinese pine |
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