CN101849505A - Method for inducing embryonal-suspensor mass (ESM) regeneration plant from immature seed of pinus massoniana - Google Patents
Method for inducing embryonal-suspensor mass (ESM) regeneration plant from immature seed of pinus massoniana Download PDFInfo
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Abstract
The invention relates to a method for inducing an embryonal-suspensor mass (ESM) regeneration plant from an immature seed of pinus massoniana. By using the characteristic of continuous schizogenesis capability of the ESM in the immature seed of the pinus massoniana, solid culture and liquid culture are performed alternatively and ESM forms a completely-grown embryo with a normal structure after a maintaining stage, a multiplication stage, an embryo pre-mature stage and a maturation culture stage, so that a pinus massoniana plant is obtained by germinating and culturing the embryo. Due to the adoption of the method, a large number of regeneration plants of a pinus massoniana embryo of which the hereditary basis is accordant can be obtained and new technology with a short period and high occurring efficiency is provided for the storage of pinus massoniana rare generic resource and the mass production of excellent-gene nursery-grown plants.
Description
One, technical field
The invention belongs to the sapling multiplication biological technical field in the forestry, be specifically related to a kind of method of inducing masson pine select tree immature seed embryo suspensor cell group (Embryonal Suspensor Mass is called for short ESM) regeneration plant.
Two, background technology
Masson pine (Pinus massoniana) is one of southern main timber tree species of China, has a very wide distribution, accumulation is big.Because its adaptability is strong, growth is fast, of many uses, afforestation is upgraded easily, cost is low, play a significantly greater role in planting trees on barren hills, aspect improving the ecological environment.Compare with northern Korean pine with the merchantable log China fir, masson pine all surpasses China fir, Korean pine on bending strength, the modulus of elasticity in static bending, compression parallel to grain, 4 indexs of end hardness; And its lumber fibre cellulose content is higher, and timber is widely used in building, sheet material, furniture, plywood, papermaking and xylem fibre industry.It is wet that masson pine timber is very water-fast, have saying of " water logging pine in thousand ", is suitable for water-bed underground engineering, is used for ore pillar and pile in a large number; The Main Ingredients and Appearance of rosin is rosin and turpentine oil, is important natural industrial chemicals, and masson pine still is the main product fat seeds of China, and the product fat phase is long, the output height, and more than the individual plant standing tree diameter of a cross-section of a tree trunk 1.3 meters above the ground 18cm, can cut fat 5～6kg every year; Masson pine timber fibre-bearing element 62% is papermaking and man-made fibres industry's important source material after the degreasing; Utilize pine stump, bending-wood can cultivate famous and precious traditional Chinese medicine Poria cocos, or the like.Therefore, strengthen protection, the significance that genetic resources of research, protection masson pine and development Masson Pine Plantation resource have to the existing standing forest of masson pine.
In the cultivation of the masson pine forest resources, adopting the plantation of genetic improvement material is one of key measure of forest culture and management success.The main at present conventional methods such as seed orchard breeding, select tree clone cottage propagation that adopt of masson pine stock breeding, reproduction speed is slower, is difficult to satisfy the demand of production application.
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 masson pine 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 maturation, sprouting and the plant regeneration of inducing, keeping of four step: ESM and propagation, body embryo, and proves that this technical system has the prospect that reaches industrialization.
Though the report that has masson pine mature seed inductor embryo to take place at present, inductivity is extremely low, is difficult to use on producing.Sino-South African Forestry University of Science and Technology once reported the rataria cultured in vitro of masson pine differing maturity, and evoked callus and body embryo take place, but do not obtain concrete result as yet.Still do not have the ESM inductor embryo that utilizes masson pine select tree immature seed both at home and abroad and take place, and realize the successful methods report of plant regeneration.
Three, summary of the invention
The objective of the invention is at current masson pine tissue culture organ difficulty takes place, shoot regeneration frequency is low, and it is extremely low with the mature zygotic embryos inductor embryo success rate of masson pine, be difficult to satisfy problems such as production application needs, the generation of the ESM inductor blast of research and utilization masson pine select tree and the new way of plant regeneration, produce large quantities of high-quality nursery stocks fast, satisfy the demand of production of forestry high-quality masson pine seedling.
The present invention is a starting material with the ESM of masson pine select tree immature seed, carrying out the multistep cultured in vitro through obtaining on the basis of repeated screening on the medium, the cleavage polyembryony generating ability of long term maintenance immature seed 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.
Technical solution of the present invention is:
A kind of method of inducing the immature seed ESM regeneration plant of masson pine select tree, it is characterized in that gathering masson pine select tree cone in the mid-June in every year, after placing one week of 4 ℃ of stored refrigerated preliminary treatment above, under aseptic condition, strip the immature embryo in the cone, cultivate by following condition of culture and priority time sequencing:
The initial of a.ESM induces
This stage minimal medium adopts the DCR medium.Under aseptic condition, the immature seed embryo is seeded in the following inducing culture, 23 ℃, secretly cultivated 40 days, induce to obtain a large amount of ESM, culture medium prescription is as follows.
KNO
3 340mg/L
Ca(NO
3)
2·4H
2O 56mg/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 27.8mg/L
H
3BO
3 6.2mg/L
MnSO
4·H
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
NiCl
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
2，4-D 2mg/L
6-BA 1mg/L
Glutamine 1g/L
Caseinhydrolysate 0.5g/L
Inositol 1g/L
Sugar 30g/L
Agar 1.9g/L
B.EMS keeps and breed
This stage minimal medium adopts the DCR medium.The EMS of institute's inducing culture gained among a. is changed under aseptic condition in the following prescription medium, under 23 ℃ of dark condition of culture, keeps and cultivated 20 days:
KNO
3 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 27.8mg/L
H
3BO
3 6.2mg/L
MnSO
4·H
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
NiCl
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
2，4-D 0.6mg/L
6-BA 0.2mg/L
Glutamine 1g/L
Caseinhydrolysate 0.5g/L
Inositol 1g/L
Sugar 30g/L
Agar 7.0g/L
The EMS that will keep cultivates after 20 days moves in the following liquid nutrient medium, 23 ℃ of dark cultivations, and shaking speed is an enrichment culture 7 days under the condition of 80～100r.p.m:
KNO
3 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
20 370mg/L
Na
2-EDTA·H
20 37.3mg/L
FeSO
4·7H
2O 27.8mg/L
H
3BO
3 6.2mg/L
MnSO
4·H
20 22.3mg/L
ZnSO
4·7H
20 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
NiCl
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
2，4-D 0.6mg/L
6-BA 0.2mg/L
Glutamine 1g/L
Caseinhydrolysate 0.5g/L
Inositol 1g/L
Sugar 30g/L
C. the prematuration period cultivation of body embryo
This stage minimal medium adopts the DCR medium.EMS after institute's enrichment culture is finished among the b. is transferred in the following prescription liquid nutrient medium, 23 ℃, the dark cultivation, shaking speed is under the condition of 80～100r.p.m, prematuration period, cultivated 7 days:
KNO
3 909.9mg/L
Ca(NO
3)
2·4H
20 236.2mg/L
NH
4NO
3 603.8mg/L
KH
2PO
4 136.1mg/L
Mg(NO
3)
2·6H
20 256.6mg/L
MgSO
4·7H
20 246.5mg/L
MgCl
2·6H
20 50.0mg/L
Na
2-EDTA·H
20 9.33mg/L
FeSO
4·7H
2O 6.95mg/L
H
3BO
3 15.5mg/L
MnSO
4·H
20 10.5mg/L
ZnSO
4·7H
20 14.4mg/L
Na
2MoO
4·2H
2O 0.125mg/L
CuSO
4·5H
2O 0.125mg/L
KI 4.15mg/L
CoCl
2 0.125mg/L
MyO-Inositol 1000mg/L
Glycine 2.0mg/L
Thiamine·HCI 1.0mg/L
Nictinic·acid 0.5mg/L
Pyridoxine·HCI 0.5mg/L
ABA 5mg/L
D. the maturation of body embryo is cultivated
This stage minimal medium adopts improvement P6 medium.With among the c. through the prematuration period material transfer of cultivating in following medium, the dark cultivation 40 days under 23 ℃ temperature:
KNO
3 909.9mg/L
Ca(NO
3)
2·4H
2O 236.2mg/L
NH
4NO
3 603.8mg/L
KH
2PO
4 136.1mg/L
Mg(NO
3)
2·6H
2O 256.6mg/L
MgSO
4·7H
2O 246.5mg/L
MgCl
2·6H
2O 50.0mg/L
Na
2-EDTA·H
2O 9.33mg/L
FeSO
4·7H
2O 6.95mg/L
H
3BO
3 15.5mg/L
MnSO
4·H
2O 10.5mg/L
ZnSO
4·7H
2O 14.4mg/L
Na
2MoO
4·2H
2O 0.125mg/L
CuSO
4·5H
2O 0.125mg/L
KI 4.15mg/L
CoCl
2 0.125mg/L
MyO-Inositol 1000mg/L
Glycine 2.0mg/L
Thiamine·HCI 1.0mg/L
Nictinic·acid 0.5mg/L
Pyridoxine·HCI 0.5mg/L
ABA 2mg/L
GA3 1mg/L
Active carbon 1g/L
PEG8000 130g/L
Sugar 25g/L
Glutamine 1g/L
Agar 7g/L
Through above-mentioned cultivation stage, body embryo completion morphology maturation and physiological ripening possess radicle, plumular axis and cotyledon, form the body embryo of structural integrity.When about 2mm left and right sides length is grown in cotyledonous body embryonic development, adopt improvement P6 minimal medium to cultivate sprouting, the regeneration that can realize plant with cultivate into seedling.
Four, 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 microphoto;
Fig. 3 is the ESM microphoto of keeping with the enrichment culture starting stage;
Fig. 4 keeps the ESM microphoto early stage with enrichment culture;
Fig. 5 is the ESM microphoto of keeping with the enrichment culture later stage;
Fig. 6 for the prematuration period the early stage body embryo microphoto cultivated;
Fig. 7 is the body embryo in the maturation;
Fig. 8 is the body embryo of growing to cotyledon period;
Fig. 9 cotyledonary embryos is sprouted
Five, embodiment
For the examination material is the immature embryo of masson pine select tree, picks up from the masson pine clone grafting seed orchard of Nanning and two places, Nan Ping, Fujian respectively, repeats for 3 year.The cone acquisition time is annual by the end of May to by the end of September, gathers once in per 10 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; Speed is transported the laboratory back to place 4 ℃ of refrigerator cold-storages to preserve standby subsequently.In 4 ℃ of at least one weeks of refrigerator preliminary treatment, help efficiently inducing of ESM.Before the inoculation, standby cone is taken out from refrigerator, with the washing lotion soaking and washing that contains washing agent, the flowing water flushing is 2 hours then.In gnotobasis, will be with seed wing, well-developed immature seed to take out in the cone, the removal seed wing with scalpel.Peel off kind of a skin with scalpel and tweezers, put 75% alcohol middling speed bubble 30 seconds, aseptic water washing 3 times was sterilized 5 minutes with 84 thimerosals of 10 times of dilutions again, and aseptic water washing 3 times places on the aseptic filter paper and blots, and is inoculated in inducing culture subsequently.
A. the embryo suspensor cell is rolled into a ball inducing culture
Inducing culture of the present invention is minimal medium with DCR, adds 2,4-D 2mg/L, 6-BA 1mg/L, glutamine 1g/L, caseinhydrolysate 0.5g/L, inositol 1g/L, sugared 30g/L, agar 7g/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 in the above-mentioned inducing culture, and visible ESM through inducing is translucent the material of shape thickness from ovule hole propagation and expand (accompanying drawing 1) at medium going out after about 20 days.Have only this present translucent, the thread ESM of thickness, could be under the medium that is fit to continuous propagation.Minority is the hard partially ESM group of faint yellow close texture and surface, all can't normally breed in successive transfer culture process subsequently by (accompanying drawing 2).
In the inducing of ESM, induction frequency and the acquisition time of immature seed embryo have bigger related.Experiment shows that being used in the masson pine immature seed embryo of being gathered between 10～June 20 June is starting material, could induce ESM propagation smoothly.
Can observe ESM from microscopically is made up of a series of embryo heads and suspensor structural material.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 (accompanying drawing 3).
, when reaching about 4mm diameter, transfer on the proliferated culture medium at the ESM culture of inducing propagation on the inducing culture.The immature seed embryo that induces ESM is transferred on the fresh inducing culture, can continue to induce the more ESM of propagation.Induce the time cycle of ESM propagation to be about 40 days, 23 ℃, the dark cultivation.
On inducing culture, induce the incubation time of EMS very crucial.If the inducing culture time is of a specified duration excessively, the EMS that induces atrophy because of the energy depletion in the medium, the EMS of translucent thickness changes opaque yellow of the harder bulk of quality or faint yellow culture into, and then loses growth vigor.
The formation and the standard recipe of DCR (Gupta and Durzan medium) minimal medium are as follows:
Macroelement
KNO
3 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
Molysite
Na
2-EDTA·H
2O 37.3mg/L
FeSO
4·7H
2O 27.8mg/L
Trace element
H
3BO
3 6.2mg/L
MnSO
4·H
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
NiCl
2 0.025mg/L
Organic substance
MyO-Inositol 200mg/L
Glycine 2.0mg/L
Thiamine·HCI 1.0mg/L
Nictinic·acid 0.5mg/L
Pyridoxine·HCI 0.5mg/L
B.EMS keeps and enrichment culture
The maintenance of EMS and proliferated culture medium, constituent is identical with the minimal medium of inducing EMS, but hormone concentration is reduced.
Masson pine ESM keep with enrichment culture be minimal medium with DCR, additional 2,4-D 0.6mg/L, 6-BA 0.2mg/L, glutamine 1g/L, caseinhydrolysate 0.5g/L, inositol 1g/L, sugared 30g/L, the solid culture medium of agar 7g/L is kept and was 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.The prescription of medium is identical with the prescription of solid multiplication medium, only removes coagulating agent agar.The initial density of cultured cell is 1.20% (V/V), and shaking speed is 80～100r.p.m, 23 ℃, the dark cultivation, and the time of enrichment culture was controlled to be about 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, through changing the successive transfer culture of culture fluid 2 to 3 times, the embryo head is spherical in shape mostly, and compact structure is grown completely, and cytoplasm is denser, and suspensor presents arrangement (accompanying drawing 5) clocklike.
C. 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, be minimal medium, remove all additional hormones, add ABA (abscisic acid) 5mg/L, stop ESM and continue schizogenesis, turn to the ripe developmental process of body embryo with P6 improvement liquid nutrient medium.Shaking speed still is controlled at about 100r.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,, can find that embryo head suspensor has all obtained good growth (accompanying drawing 6) 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.
Improve the formation of P6 medium and fill a prescription as follows:
Macroelement
KNO
3 909.9mg/L
Ca(NO
3)
2·4H
2O 236.2mg/L
NH
4NO
3 603.8mg/L
KH
2PO
4 136.1mg/L
Mg(NO
3)
2·6H
2O 256.6mg/L
MgSO
4·7H
2O 246.5mg/L
MgCl
2·6H
2O 50.0mg/L
Molysite
Na
2-EDTA·H
2O 9.33mg/L
FeSO
4·7H
2O 6.95mg/L
Trace element
H
3BO
3 15.5mg/L
MnSO
4·H
2O 10.5mg/L
ZnSO
4·7H
2O 14.4mg/L
Na
2MoO
4·2H
2O 0.125mg/L
CuSO
4·5H
2O 0.125mg/L
KI 4.15mg/L
CoCl
2 0.125mg/L
Organic substance
MyO-Inositol 1000mg/L
Glycine 2.0mg/L
Thiamine·HCI 1.0mg/L
Nictinic·acid 0.5mg/L
Pyridoxine·HCI 0.5mg/L
D. the 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, with the cotyledonary embryos early stage similar (accompanying drawing 5) in the zygotic embryo growth course, the body embryo of this state, grow success rate for the regular embryo in cultivating in the later stage than higher.Minority body embryo is also arranged after pre-maturation is finished, the embryo head does not become and polishes greatly, and the suspensor deformity that shortens disappears even, and this state can't be grown the organizator embryo in the later stage cultivation, perhaps can only form the body embryo of deformity, rest on the column embryo and no longer continue development growth.
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 P6 improvement minimal medium, additional 2mg/L ABA and 3mg/L gibberellin GA3, active carbon 1g/L, agar 7g/L.In order to regulate osmotic pressure, add PEG (polyethylene glycol) 8000 130g/L, add the sugared 25g/L of SILVER REAGENT purity in addition, glutamine 1g/L.
In this stage, the regulating action of osmotic pressure is very obvious.Sucrose, salt, polyethylene glycol (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 active carbon 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 active carbon, 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 40 days (23 ℃, the dark cultivation).
Through the cultivation of a wheat harvesting period, body embryo completion morphology maturation and physiological ripening form the complete embryonic knob structure that possesses radicle, plumular axis and cotyledon.Under suitable condition of culture, the embryo in this stage has sprouting ability and the final form generating ability that produces whole plant.
When cotyledonary embryos grows into general 2mm left and right sides length, adopt conventional method to cultivate and sprout, the regeneration that realizes plant with cultivate into seedling.
Beneficial effect:
Advantage of the present invention is that breeding coefficient height, breeding are not subjected to the restriction in season, for the Fast-propagation of masson pine genetic improvement material provides a kind of high effective way, not only be conducive to the preservation of masson pine genetic resources, and for the growth of accelerating the masson pine excellent material, a large amount of planting materials through genetic improvement is provided, alleviates society to the urgent demand of masson pine genetic improvement nursery stock, provide a kind of cycle short, the efficient height, the nursery stock production technology that cost is low.
Claims (1)
1. method of inducing embryonal-suspensor mass (ESM) regeneration plant from immature seed of pinus massoniana, it is characterized in that gathering masson pine select tree cone in the mid-June in every year, after placing one week of 4 ℃ of stored refrigerated preliminary treatment above, under aseptic condition, strip the immature embryo in the cone, cultivate by following condition of culture and priority time sequencing:
The initial of a.ESM induces
Under aseptic condition the immature seed embryo is seeded in the following inducing culture, the dark cultivation 40 days under 23 ℃ temperature induces to obtain a large amount of ESM, and culture medium prescription is as follows:
KNO
3 340mg/L
Ca(NO
3)
2·4H
2O 56mg/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 27.8mg/L
H
3BO
3 6.2mg/L
MnSO
4·H
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
NiCl
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
2，4-D 2mg/L
6-BA 1mg/L
Glutamine 1g/L
Caseinhydrolysate 0.5g/L
Inositol 1g/L
Sugar 30g/L
Agar 1.9g/L
B.EMS keeps and breed
The EMS of institute's inducing culture gained among a. is changed under aseptic condition in the following prescription medium, under 23 ℃, dark condition of culture, keeps and cultivated 20 days:
KNO
3 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 27.8mg/L
H
3BO
3 6.2mg/L
MnSO
4·H
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
NiCl
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
2，4-D 0.6mg/L
6-BA 0.2mg/L
Glutamine 1g/L
Caseinhydrolysate 0.5g/L
Inositol 1g/L
Sugar 30g/L
Agar 7.0g/L
The EMS that will keep cultivates after 20 days moves in the following liquid nutrient medium, and 23 ℃, dark cultivation, shaking speed is an enrichment culture 7 days under the condition of 80～100r.p.m:
KNO
3 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 27.8mg/L
H
3BO
3 6.2mg/L
MnSO
4·H
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
NiCl
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
2，4-D 0.6mg/L
6-BA 0.2mg/L
Glutamine 1g/L
Caseinhydrolysate 0.5g/L
Inositol 1g/L
Sugar 30g/L
C. the prematuration period cultivation of body embryo
EMS after institute's enrichment culture is finished among the b. is transferred in the following prescription liquid nutrient medium, and 23 ℃, the dark cultivation, shaking speed is under the condition of 80～100r.p.m, and prematuration period cultivated 7 days:
KNO
3 909.9mg/L
Ca(NO
3)
2·4H
2O 236.2mg/L
NH
4NO
3 603.8mg/L
KH
2PO
4 136.1mg/L
Mg(NO
3)
2·6H
2O 256.6mg/L
MgSO
4·7H
2O 246.5mg/L
MgCl
2·6H
2O 50.0mg/L
Na
2-EDTA·H
2O 9.33mg/L
FeSO
4·7H
2O 6.95mg/L
H
3BO
3 15.5mg/L
MnSO
4·H
2O 10.5mg/L
ZnSO
4·7H
2O 14.4mg/L
Na
2MoO
4·2H
2O 0.125mg/L
CuSO
4·5H
2O 0.125mg/L
KI 4.15mg/L
CoCl
2 0.125mg/L
MyO-Inositol 1000mg/L
Glycine 2.0mg/L
Thiamine·HCI 1.0mg/L
Nictinic·acid 0.5mg/L
Pyridoxine·HCI 0.5mg/L
ABA 5mg/L
D. the maturation of body embryo is cultivated
With among the c. through the prematuration period material transfer of cultivating in following medium, the dark cultivation 40 days under 23 ℃ temperature:
KNO
3 909.9mg/L
Ca(NO
3)
2·4H
2O 236.2mg/L
NH
4NO
3 603.8mg/L
KH
2PO
4 136.1mg/L
Mg(NO
3)
2·6H
2O 256.6mg/L
MgSO
4·7H
2O 246.5mg/L
MgCl
2·6H
2O 50.0mg/L
Na
2-EDTA·H
2O 9.33mg/L
FeSO
4·7H
2O 6.95mg/L
H
3BO
3 15.5mg/L
MnSO
4·H
2O 10.5mg/L
ZnSO
4·7H
2O 14.4mg/L
Na
2MoO
4·2H
2O 0.125mg/L
CuSO
4·5H
2O 0.125mg/L
KI 4.15mg/L
CoCl
2 0.125mg/L
MyO-Inositol 1000mg/L
Glycine 2.0mg/L
Thiamine·HCI 1.0mg/L
Nictinic·acid 0.5mg/L
Pyridoxine·HCI 0.5mg/L
ABA 2mg/L
GA3 1mg/L
Active carbon 1g/L
PEG8000 130g/L
Sugar 25g/L
Glutamine 1g/L
Agar 7g/L
When about 2mm left and right sides length is grown in cotyledonous body embryonic development, adopt improvement P6 minimal medium to cultivate sprouting, the regeneration that can realize plant with cultivate into seedling.
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| CN110305833A (en) * | 2019-07-10 | 2019-10-08 | 广西壮族自治区林业科学研究院 | A kind of suspension culture method of masson pine cells,primordial proliferation |
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| CN114788496A (en) * | 2022-04-07 | 2022-07-26 | 江苏省中国科学院植物研究所 | Method for inducing efficient somatic embryogenesis of larch through solid-liquid alternate culture |
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