CN101983556A - Spruce somatic embryogenesis and plant regeneration method - Google Patents
Spruce somatic embryogenesis and plant regeneration method Download PDFInfo
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Abstract
The invention discloses a spruce somatic embryogenesis and plant regeneration method, which comprises preparation of immature seed embryo of spruce, initial induction of embryonal suspensor mass (ESM), maintenance and proliferation of the ESM, prophase maturation culture of the somatic embryo, maturation culture of the somatic embryo and germination of the somatic. The spruce plant regeneration method has the advantages of high propagation coefficient and no limitation on the propagation season and provides an efficient means for the rapid propagation of the genetically improved materials of spruce, which not only is conducive to the storage of genetic resources of spruce, but also provides a large number of genetically improved plant materials for accelerating the cultivation of the improved materials of spruce, eases urgent requirements of the society on the genetically improved nursery stock of spruce, and provides a technology for producing an improved variety of nursery stock with short period, high efficiency and low cost.
Description
Technical field
The present invention relates to the sapling multiplication biological technical field in the forestry, be specifically related to a kind of (Embryonal Suspensor Mass of immature zygotic embryos embryo suspensor cell group that induces the hybrid combination of China fir select tree, be called for short ESM) vitro proliferation, develop into the method for complete body blast and regeneration plant.
Background technology
China fir (Cunninghamia lanceolata (Lamb.) Hook) is subordinate to Taxodiaceae (Taxodiaceae) Cunninghamia (Cunninghamia), is one of distinctive evergreen coniferous species of China.The China fir growth is fast, and form is perfectly straight satisfactory, and again with good wood property, the grain of wood attractive in appearance is celebrated, and purposes is very extensive, is China's important commodity material reproducting tree species.Since generation nineteen sixty, the research of China fir fine-variety breeding has been cultivated large quantities of fine provenances, family and choiceness and has been used for afforestation, builds for China fast-growing and high-yield plantation and has made significant contribution.But owing to increasing sharply of afforestation area, the demand to the excellent Chinese fir seedling in the society is very urgent.Yet owing to be subjected to the harm of the real insect of climatic anomaly and China fir kind during the nearly last ten years, and the seed production in Chinese fir seeds garden, the phenomenon of biennial bearing is serious, breeding seed production and sowing quality instability.China fir breeding seedling under-supply seriously restricting the development of Chinese Fir Plantation woods.Therefore, be necessary to research and develop the asexual quick breeding of excellent Chinese fir germ plasm resource.Advantages such as it is fast that the somatic embryo generation technique has reproduction speed, and cultivation cycle is short, and production cost is low, this provides technical support for the breed bottleneck and commercialization and batch production production that solve the excellent Chinese fir seedling.
The generation of angiosperm body embryo and the thought of plant regeneration, the plant cell that comes from Haberlandt proposition in 1902 has totipotent notion, and promptly each cell of plant corpus all has new individual potential ability of bud into.Under the artificial culture condition, plant soma can be induced the embryo shape structure (embryoid) that is differentiated to form as the sexual reproduction zygotic embryo, and then forms artificial seed or the complete plant of formation that directly regenerates by the cover artificial endosperm.Because all there is the possibility of inducing into embryo theoretically in each somatic cell of plant, and reproduction speed soon, is not subjected to the restriction of natural conditions such as area, season and disastrous weather; For woody plant, needn't wait for very long sexual generation, also needn't as conventional vegetative propagation, need sufficient rudiment to transplant a cutting, in case obtain excellent material, just can breed good seed than the speed of routine breeding fast tens of times even hundreds of times.
As China fir gymnospermous, the generation of body embryo and angiospermous somatic embryo have diverse approach and mechanism, ripe seed and the plant that comes by seed development, and it is very difficult that trophozyte changes cells,primordial into.But in coniferous species, the egagametophyte after fertilization develops into that some nucellar cell is in the undifferentiated stage in the process of seed, under suitable condition of culture, can utilize the cleavage polyembryony characteristic, the inductor embryogenesis.Gupta etc. (1985,1995) reported and utilized this biological property, induce the body embryo of pesudotsuga taxifolia (Pseudotsuga menziesii) and wet-land pine tree (Pinus elliottii), it roughly can be divided into four steps: embryo suspensor cell group (ESM:Embryogenesis suspensor mass) induces, keeps and maturation, sprouting and the plant regeneration of propagation, body embryo, and proves that this technical system has the prospect that reaches industrialization.
Though the report that has the hypocotyl inductor embryo after the China fir mature seed is sprouted to take place at present, can only on explant, form indivedual somatic embryos, there is the problem that inductivity is low, quantity is few, be difficult on producing, use.Still does not have both at home and abroad and utilize China fir select tree immature seed to induce ESM, this indirect body embryogenesis path takes place and realize that the successful methods of plant regeneration reports by liquid propagation ESM and inductor embryo.
Summary of the invention
Goal of the invention: the objective of the invention is at current China fir tissue culture organ take place difficulty, shoot regeneration frequency low, be difficult to satisfy problem such as production application needs, provide a kind of immature embryo of China fir select tree that utilizes to induce embryonal suspensor mass to form, and then realize that somatic embryo takes place and the method for regeneration China fir plant, can produce large quantities of high-quality nursery stocks fast in the hope of realizing, satisfy the demand of production of forestry high-quality China fir seedling.
Technical scheme: in order to realize the foregoing invention purpose, the technical solution used in the present invention is as follows:
The present invention is a starting material with China fir select tree immature embryo, on the suitable culture base, carry out the multistep cultured in vitro, the cleavage polyembryony generating ability of long term maintenance immature embryo ESM, through embryo callus subculture induce, keep, propagation, body embryo the prematuration period and the stage such as maturation cultivate, obtain the body embryo, cultivate the regeneration that realizes plant through sprouting again.
A kind of China fir somatic embryo takes place and plant regeneration method, may further comprise the steps:
(1) preparation of China fir immature seed embryo
Before and after annual mid-March, select to show good parent configuration hybrid combination through progeny testing.And, gather the cone of China fir hybrid combination mid-June, under anatomical lens, observe the developmental process of China fir zygotic embryo.Get and grow, place one week of stored refrigerated under 4 ℃ of conditions rapidly to embryo's selection, advantage embryo, column embryo and the China fir cone in early stage cotyledonary embryos stage.Under aseptic condition, the immature seed embryo that strips in the cone is standby; Because the climatic region difference of different China firs growth and the variation of meteorological condition between year begin to be suitable cone sample time the choice phase with observed embryo under the anatomical lens till the early stage cotyledonary embryos stage.
(2) the initial of ESM induces
Initial induction period adopts the basic cultivation of Gupta and Durzan minimal medium (DCR).Under aseptic condition, the immature seed embryo is seeded to following inducing in the solid culture medium, 8 seed embryos of each culture dish inoculation, 23 ℃, secretly cultivated 13～18 days, just can induce a large amount of ESM of acquisition; Induce the solid culture based formulas as follows: DCR minimal medium, additional 2,4 dichlorophenoxyacetic acid (2,4-D) 2～6mg/L, 6-benzyl aminoadenine (6-BA) 0.5～1mg/L, 6-furans aminopurine (KT) 0.5mg/L, vitamin C (Vc) 10mg/L, L-glutaminate (L-Gln) 0.45g/L, caseinhydrolysate 0.5g/L, inositol 0.1g/L, maltose 15g/L, active carbon (Ac) 2.5g/L, quartzy agar 2.3g/L.
(3) EMS's keeps and breed
Keep with the multiplicative stage minimal medium and adopt the DCR medium.Under aseptic condition, the EMS of inducing culture gained in (2) changed over to keep in the solid culture medium, 23 ℃, secretly cultivated 20 days;
The prescription of keeping solid culture medium is: the DCR minimal medium, add 2,4-D 0.5-2mg/L, 6-BA0.2-0.5mg/L, KT 0.5mg/L, Vc 10mg/L, glutamine 0.45g/L, caseinhydrolysate 0.5g/L, inositol 0.1g/L, maltose 20-25g/L, active carbon (Ac) 2.5g/L, quartzy agar 2.5g/L.
Under aseptic condition, move in the liquid proliferated culture medium keeping the EMS of cultivation after 20 days, 23 ℃ of dark cultivations, shaking speed is 85-90r.p.m, enrichment culture 7 days; Liquid enrichment culture based formulas is: the DCR minimal medium, add 2,4-D 0.5-2mg/L, 6-BA 0.2mg/L, Vc 10mg/L, glutamine 0.45g/L, caseinhydrolysate 0.5g/L, inositol 2g/L, maltose 30g/L.
(4) the prematuration period cultivation of body embryo
The prematuration period cultivation stage minimal medium adopt the DCR medium.In the liquid nutrient medium, 23 ℃, shaking speed is 85r.p.m, secretly cultivates 7 days prematuration period that EMS after institute's enrichment culture is finished in (3) being transferred to, and can obtain the comparatively fine and close EMS in surface; The prematuration period liquid culture based formulas be: DCR minimal medium, additional abscisic acid (ABA) 1,5 or 10mg/L, gibberellin (GA) 0.1-1mg/L, Vc 10mg/L, glutamine 0.45g/L, caseinhydrolysate 0.5g/L, inositol 5g/L, maltose 30g/L.
(5) maturation of body embryo is cultivated
Ripe cultivation stage minimal medium adopts the DCR medium.With in (4) through the prematuration period material transfer of cultivating in maturation medium, 23 ℃, secretly cultivated 50 days.The maturation medium prescription is: DCR minimal medium, additional ABA 3-8mg/L, GA 1-5mg/L, Vc 10mg/L, polyethylene glycol (PEG 8000) 100-200g/L, glutamine 0.45g/L, L-proline (L-Pro) 0.2g/L, L-aspartic acid (L-Asp) 0.2g/L, caseinhydrolysate 0.5g/L, inositol 5g/L, maltose 30g/L, Ac 2.5g/L, quartzy agar 2.5g/L.
(6) sprouting of body embryo
Through above-mentioned cultivation stage, naked eyes promptly can be observed the column embryo of growth, when the column embryonic development when being about cotyledonary embryos long about 4～5mm, under aseptic condition, be transferred on the solid DCR minimal medium, further cultivate, until sprouting, the regeneration that can realize plant with cultivate into seedling.
Beneficial effect: advantage of the present invention is that reproduction coefficient height, breeding are not subjected to the restriction in season, for the quick breeding of China fir genetic improvement material provides a kind of efficient approach, not only help the preservation of China fir genetic resources, and for quickening breeding of excellent Chinese fir material, a large amount of planting materials through genetic improvement is provided, alleviates the urgent demand of society, provide a kind of cycle short China fir genetic improvement nursery stock, the efficient height, the nursery stock production technology that cost is low.
Description of drawings
Fig. 1 is the embryo suspensor cell group microphoto of inducing the starting stage.
Fig. 2 induces the non-embryo suspensor cell of starting stage to roll into a ball photo.
Fig. 3 is the ESM photo of keeping with the enrichment culture starting stage.
Fig. 4 is the liquid culture photo of keeping with the enrichment culture later stage.
Fig. 5 keeps the ESM microphoto early stage with enrichment culture.
Fig. 6 is the ESM microphoto of keeping with the enrichment culture later stage.
The early stage somatic embryo microphoto of cultivating the prematuration period that Fig. 7 being.
Fig. 8 is the body embryo photo that the column embryo is grown to cotyledonary embryos in the maturation.
Fig. 9 is the body embryo photo of growing in the maturation to cotyledon period.
Figure 10 is a cotyledon embryo germination photo in the maturation.
Figure 11, Figure 12, Figure 13 and Figure 14 be respectively the body embryo germination body embryo seedling of taking root, growing true leaf.。。The photo in stage.
Embodiment
The present invention is described further below in conjunction with drawings and Examples.
Embodiment 1
(1) preparation of China fir immature seed embryo
For the examination material is the immature embryo of China fir select tree hybrid combination, picks up from the China fir clonal seed orchard of Shunchang, Fujian and Wei Min two places respectively, 2 year of repeated sampling.The cone acquisition time is annual by the end of June to by the end of August, gathers once in per 7 days.The corresponding seed development stage of each seed collecting time point is respectively rataria to mature embryo developmental stage process.After cone was gathered, put in the good ice chest of pre-freeze at the scene, and moistening gauze parcel prevents dehydration; It is standby that speed places 4 ℃ of refrigerator cold-storages preservations subsequently.4 ℃ of refrigerator cold-storages are preserved at least one week, help efficiently inducing of ESM.Before the inoculation, standby cone is taken out from refrigerator, take out in the cone with scalpel and tweezers and be with seed wing, well-developed immature seed, with the liquid detergent that is rich in efficient composite reactive agent, for example, carving board liquid detergent, be made into the washing lotion soaking and washing 10min of 1: 50 concentration, then flowing water flushing 30min.In gnotobasis, will transfer in the autoclaved triangular flask through the preliminary immature seed that cleans, 75% alcohol middling speed bubble 30 seconds is used 0.1%HgCl again
2Sterilization 9min, use aseptic water washing at last 3 times, place on the aseptic filter paper and blot, remove kind of a skin with scalpel and tweezers subsequently, be inoculated in inducing culture, 8 seeds of each culture dish inoculation, general each processing can be inoculated 20 to 30 culture dishes, each genotype is prepared 10 cones, has 12 genotype, and full seed quantity has than big difference because of genotype.
(2) embryo suspensor cell group inducing culture
The solid culture medium of inducing of the present invention is minimal medium with DCR, adds 2,4-D 2-6mg/L, 6-BA0.5mg/L, KT 0.5mg/L, glutamine 0.45g/L, caseinhydrolysate 0.5g/L, inositol 0.1g/L, maltose 15g/L, quartzy agar 2.3g/L.Wherein, 2 of additional higher concentration, the 4-D purpose is to improve the inductivity of embryo suspensor cell group.
Immature embryo is seeded to above-mentioned inducing in the solid culture medium, 23 ℃ of temperature controls, secretly cultivated 13～18 days, as seen the ESM through inducing is translucent the high material of shape water content from ovule hole propagation and expand at medium after going out, as shown in Figure 1, have only this present translucent, the ESM of the barbed shape of thickness could constantly breed and keep original embryo under the medium that is fit to.The minority close texture that is creamy white, there is transparent filament on the surface, and viscosity is big, and the rapid ESM group of growing all can't keep embryo as shown in Figure 2 for a long time in successive transfer culture process subsequently, finally all form the maxicell of the big vacuole of tool.
In the inducing of ESM, the acquisition time of induction frequency and immature seed embryo has substantial connection.Test shows, with the China fir immature seed embryo of being gathered between 10～August 10 July is starting material, can induce ESM propagation smoothly, have only few part can induce ESM early than the immature seed at the beginning of 7 months, and the easy precocity of the immature seed in later stage be sprouted into seedling.
Can observe ESM from microscopically is made up of a series of embryo heads and suspensor structural material.As shown in Figure 3, the embryo head part is smaller, has cytoplasm, is connecting the transparent or translucent suspensor of long height vacuolization.Whole embryo is made of translucent loose suspensor bundle and the irregular embryo of profile top.A series of embryo suspensor cells have just constituted embryo suspensor cell group.
The formation and the standard recipe of DCR (Gupta and Durzan medium) minimal medium are as follows:
KNO3 340mg/L
Ca(NO
3)
2·4H
2O 556mg/L
NH
4NO
3 400mg/L
KH
2PO
4 170mg/L
CaCl
2·2H
2O 85mg/L
MgSO
4·7H
2O 370mg/L
Na
2-EDTA·H
2O 37.3mg/L
FeSO
4·7H
2O 27.9mg/L
H
3BO
3 6.2mg/L
MnSO
4·4H
2O 22.3mg/L
ZnSO
4·7H
2O 8.6mg/L
Na
2MoO
4·2H
2O 0.25mg/L
CuSO
4·5H
2O 0.25mg/L
KI 0.83mg/L
CoCl
2 0.025mg/L
MyO-Inositol 200mg/L
Glycine 2.0mg/L
Thiamine·HCI 1.0mg/L
Nictinic·acid 0.5mg/L
Pyridoxine·HCI 0.5mg/L
(3) EMS's keeps and enrichment culture
EMS keeps and proliferated culture medium, and constituent is identical with the minimal medium of inducing EMS, but hormone concentration is reduced.China fir ESM keep with enrichment culture be minimal medium with DCR, additional 2,4-D1mg/L, 6-BA 0.5mg/L, KT 0.5mg/L, glutamine 0.45g/L, caseinhydrolysate 0.5g/L, inositol 0.1g/L, sugar 20-25g/L, Ac 2.5g/L, the solid culture medium of quartzy agar 2.5g/L, keep and cultivated 20 days, 23 ℃, the dark cultivation; Use the liquid nutrient medium of DCR then, carry out the propagation of ESM as the minimal medium constituent.Medium supplemented 2,4-D 1mg/L, 6-BA 0.2mg/L, glutamine 0.45g/L, caseinhydrolysate 0.5g/L, inositol 2g/L, maltose 30g/L.The initial density of cultured cell is 1.20% (V/V), and shaking speed is 85～90r.p.m, 23 ℃, the dark cultivation, and the time of enrichment culture was controlled to be about 7 days, as shown in Figure 4, for cultivating the EMS growth conditions picture after 7 days.
Keep and the multiplicative stage, the solid culture main purpose is to keep the embryo and the enlarged culture thing radix of callus; The purpose of liquid culture is to improve the growth rate of ESM.In the liquid culture stage, strict shaking speed and enrichment culture time are controlled within the prescribed limit.
Under the liquid culture condition, can be through changing the successive transfer culture of culture fluid 2 to 3 times, as shown in Figure 5 and Figure 6, the embryo head is spherical in shape mostly, and compact structure is grown completely, and cytoplasm is denser, and suspensor presents arrangement clocklike.
(4) the prematuration period cultivation of body embryo
The prematuration period key technique cultivated of body embryo is to stop the continuation schizogenesis of ESM and regulate the synchronization that the embryo culture materials is grown.Through after the enrichment culture, the growth of ESM is in asynchronous state.Most of cleavage polyembryony is grown very fast, enters the cotyledonary embryos preliminary stage, even and still some ESM rest on the proembryo stage.The synchronism of body embryonic development is the key of inductor embryo normal development in the later stage incubation.
In the synchronization incubation of body embryo, liquid nutrient medium is a minimal medium with DCR, removes all additional hormones, adds ABA 1,5,8 or 10mg/L, stops ESM and continues schizogenesis, turns to the ripe developmental process of body embryo.Shaking speed still is controlled at about 85r.p.m, 23 ℃, secretly cultivate about 7 days.
ABA has key effect in the synchronously regulating and controlling that promotes coniferous tree body embryo, can obviously promote the maturation of body embryo, suppresses the developmental process of precocious body embryo, reduces the occurrence frequency of abnormal embryo.ABA also helps promoting simultaneously the accumulation of reserve substance in the body embryo.After handling through ABA,, as shown in Figure 6, can find that embryo head suspensor has all obtained good growth by microexamination, the embryo head since the accumulation of reserve substance and fine and close more smooth, suspensor is longer, and synchronization has had good improvement.
(5) maturation of body embryo is cultivated
The maturation of body embryo is with all multifactor relevant: at first, relevant with the development condition of its early stage body embryo.Early stage body growth of the embryo state can be judged by microexamination.It is big that embryo capitulum compact structure, volume become, simultaneously suspensor cell become slender closely, similar early stage with the cotyledonary embryos in the zygotic embryo growth course, the body embryo of this state is grown success rate for the regular embryo than higher in cultivating in the later stage.Secondly, the maturation of body embryo is also relevant with the cultivation microenvironment with medium.
The maturation of body embryo is cultivated in the present embodiment, adopts the DCR minimal medium, additional ABA 5mg/L and gibberellin GA3mg/L, Ac 2g/L, quartzy agar 2.5g/L.In order to regulate osmotic pressure, add PEG8000150g/L, add maltose 20g/L in addition, glutamine 0.45g/L, hydrolysis network albumen 0.5g/L, proline 0.2g/L, aspartic acid 0.2g/L.
In this stage, the regulating action of osmotic pressure is very obvious.Sucrose, salt, PEG, mannitol etc. can be as the material of regulating osmotic pressure.But they regulate the mechanism difference of osmotic pressure.Cell has the active transport ability to materials such as sugar, salt, mannitol.Permeate through cell membranes such as sugar, salt, mannitol and cell wall enter in the cell, change the osmotic pressure of cell.PEG is as a kind of macromolecule osmoticum, and itself can not infiltrate living cells, but regulates the osmotic pressure of cell by the mode of water stress, reduces the moisture in the body embryo.PEG and sugar have synergy, and both can influence the growth of body embryo jointly by adjusting cell permeability of the membrane and physical state.Comparatively speaking, PEG and sugar are used in combination, and more help the growth of body embryo.Add certain density Ac in the medium, the differentiation and the growth of somatic embryo had certain facilitation.Use ABA can not suppress the growth of precocious cotyledonary embryos separately, only in containing the medium of ABA, add Ac, could suppress the continuation of precocious cotyledonary embryos and grow, improve the growth of cotyledonary embryos apical meristem, make in its form and the physiological growth more near zygotic embryo.The ripe incubation time of body embryo is controlled to be 30 days, and 23 ℃, the dark cultivation.Through nearly bimestrial cultivation, shown in Fig. 7,8,9 and 10, be respectively the ripe growth conditions of cultivating different phase, body embryo completion morphology maturation and physiological ripening form the complete embryo shape structure that possesses radicle, plumular axis and cotyledon.When being fully-developed body embryo, when basic carbon source was provided, the body embryo had sprouting ability and the final form generating ability that produces whole plant.
(6) sprouting of body embryo
When cotyledon embryonic development during to general 4～5mm left and right sides length, adopt conventional method to cultivate and sprout, the regeneration that realizes China fir with cultivate into seedling.Shown in Figure 11,12,13 and 14, for China fir is cultivated into the seedling growth conditions.
Embodiment 2
In the step (3), keep 2 in the solid culture based formulas, 4-D is 2mg/L, and 6-BA is 0.5mg/L, 2 in the liquid enrichment culture based formulas, and 4-D is 0.5mg/L; In the step (5), the ABA in the maturation medium prescription is 8mg/L, and GA is 1mg/L, and PEG 8000 is 200g/L.Other each reactions steps and condition are all with embodiment 1, and result of the test shows, can reach the effect of embodiment 1.
Embodiment 3
In the step (3), keep 2 in the solid culture based formulas, 4-D is 0.5mg/L, and 6-BA is 0.2mg/L, 2 in the liquid enrichment culture based formulas, and 4-D is 2mg/L; In the step (5), the ABA in the maturation medium prescription is 3mg/L, and GA is 5mg/L, and PEG 8000 is 100g/L.Other each reactions steps and condition are all with embodiment 1, and result of the test shows that result of the test shows, can reach the effect of embodiment 1.
Claims (1)
1. a China fir somatic embryo takes place and plant regeneration method, it is characterized in that, may further comprise the steps:
(1) preparation of China fir immature seed embryo
Gather the cone of China fir hybrid combination, under anatomical lens, observe the China fir zygotic embryo, get and grow, place one week of stored refrigerated under 4 ℃ of conditions rapidly to embryo's selection, advantage embryo, column embryo and the China fir cone in early stage cotyledonary embryos stage.Under aseptic condition, the immature seed embryo that strips in the cone is standby;
(2) the initial of ESM induces
Under aseptic condition, the immature seed embryo is seeded to induces in the solid culture medium, 8 seed embryos of each culture dish inoculation, were secretly cultivated 13～18 days 23 ℃ of temperature controls, induced to obtain ESM; Wherein, induce the prescription of solid culture medium to be: DCR is a minimal medium, additional 2,4-D 2～6mg/L, 6-BA 0.5～1.0mg/L, KT 0.5mg/L, Vc 10mg/L, L-Gln 0.45g/L, caseinhydrolysate 0.5g/L, inositol 0.1g/L, maltose 15g/L, Ac 2.5g/L, quartzy agar 2.3g/L;
(3) EMS's keeps and breed
Under aseptic condition, the EMS that induces acquisition in the step (2) changed over to keep in the solid culture medium, 23 ℃ of temperature controls were secretly cultivated 20 days; Wherein, the prescription of keeping solid culture medium of EMS is: DCR minimal medium, 2,4-D0.5-2mg/L, 6-BA 0.2-0.5mg/L, KT 0.5mg/L, Vc 10mg/L, glutamine 0.45g/L, caseinhydrolysate 0.5g/L, inositol 0.1g/L, maltose 20-25g/L, Ac 2.5g/L, quartzy agar 2.5g/L;
Move into the liquid proliferated culture medium with keeping the EMS of cultivation after 20 days, 23 ℃ of temperature controls, shaking speed is 85～90r.p.m, secretly cultivates 7 days; Wherein, the prescription of liquid proliferated culture medium is: DCR minimal medium, 2,4-D0.5-2mg/L, 6-BA 0.2mg/L, Ac 10mg/L, glutamine 0.45g/L, caseinhydrolysate 0.5g/L, inositol 2g/L, maltose 30g/L;
(4) the prematuration period cultivation of body embryo
Under aseptic condition, with in the step (3) prematuration period that the EMS of enrichment culture transfers in the liquid nutrient medium, 23 ℃ of temperature controls, shaking speed is 85r.p.m, secretly cultivates 7 days; Wherein, the liquid culture based formulas is the prematuration period: DCR minimal medium, ABA 5-10mg/L, GA 0.1～1.0mg/L, Vc 10mg/L, glutamine 0.45g/L, caseinhydrolysate 0.5g/L, inositol 5g/L, maltose 30g/L;
(5) maturation of body embryo is cultivated
Under aseptic condition, with in the step (4) through the prematuration period material transfer of cultivating in maturation medium, 23 ℃ of temperature controls, secretly cultivate 30 days after, turning out length is the cotyledonary embryos of 4～5mm; Wherein, the prescription of maturation medium is: DCR minimal medium, ABA 3-8mg/L, GA1-5mg/L, Vc 10mg/L, PEG 8000 100-200g/L, glutamine 0.45g/L, L-Pro 0.2g/L, L-Asp 0.2g/L, caseinhydrolysate 0.5g/L, inositol 5g/L, maltose 30g/L, active carbon 2.5g/L, quartzy agar 2.5g/L;
(6) sprouting of body embryo
Under aseptic condition, be that the cotyledonary embryos of 4～5mm is transferred to solid DCR minimal medium with length, the conventional cultivation until sprouting, the regeneration that can realize plant with cultivate into seedling.
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