CN101240260B

CN101240260B - Low density spreading methods for somatic embryogenesis

Info

Publication number: CN101240260B
Application number: CN2007101618410A
Authority: CN
Inventors: 詹姆斯·A·格罗布; 斯特凡妮·A·布鲁西格
Original assignee: Weyerhaeuser Co
Current assignee: Weyerhaeuser Co
Priority date: 2006-09-28
Filing date: 2007-09-24
Publication date: 2011-11-16
Anticipated expiration: 2027-09-24
Also published as: AR062905A1; FI20070735A0; SE531226C2; BRPI0704031B1; CA2604700C; FI20070735A; BRPI0704031A2; AU2007216903B2; AU2007216903A1; FI121727B; UY30608A1; CN101240260A; SE0702034L; NZ561856A; CA2604700A1

Abstract

In one aspect, the present invention provides methods of producing conifer cotyledonary somatic embryos from pre-cotyledonary embryos. The methods of this aspect of the invention include the step of (a) dispensing a plurality of pre-cotyledonary embryos onto a porous material horizontally disposed over a non-porous surface in a volume of sterile dilution medium sufficient to submerge at least the surface of the porous material, thereby uniformly dispersing the pre-cotyledonary embryos; (b) removing the sterile dilution medium from the non-absorbent porous material, thereby trapping the uniformly dispersed pre-cotyledonary embryos on the porous material; and (c) contacting the pre-cotyledonary embryos trapped on the porous material with development medium for a period of time sufficient to produce conifer cotyledonary somatic embryos.

Description

Be used for the low density spreading methods that somatic embryo takes place

Invention field

The present invention relates to the method for produced in vitro plant embryos, and optional method of producing plant from plant embryos.

Background of invention

In order to make timber-work, to softwood tree, such as the demand sustainable growth of pine tree and fir.For providing ample supply acerose problem, the solution of a suggestion is the individual softwood tree that identification has required feature, as fast growth, and the identical clone of numerous heredity of producing excellent tree by somatic cell clone.

Somatic cell clone is a method of creating the identical tree of heredity from the somatic tissue of tree.The somatic tissue of tree is different from male and tree tissue female gamete.In a kind of method of somatic cell clone, tree somatic tissue cultivates and is comprising hormone, and in the initial substratum such as plant hormone and/or phytokinin, described hormone starts and forms the embryogenesis cells that can develop into somatic embryo.Then, embryogenesis cells is further cultivated in keeping substratum, and this substratum promotes the propagation of embryogenesis cells, cotyledon embryo (that is the embryo who, does not have cotyledon) before forming.The embryogenesis cells of propagation is cultivated in growing substratum then, and this substratum promotes the growth and the maturation of cotyledon somatic embryo, and this embryo for example can be positioned in the synthetic seed and be seeded in soil, and germinateing generates the softwood tree seedling.The seedling portable is to continued growth vegetatively, and final results generate timber or timber derived product.Perhaps, the cotyledon somatic embryo also can germinate in the germination substratum, is transferred to the soil further growth thereafter.

Somatic cell clone softwood tree embryo's lasting problem is to stimulate efficient and cost to form the somatic embryo of the generation plant that can germinate effectively.In the outer somatic embryos of coniferous trees tire that forms of preferred body and the softwood tree seed in the body softwood tree zygotic embryo of formation similar or identical on physics and physiology.Therefore, to from the softwood tree embryogenesis cells, producing the method for viable somatic embryos of coniferous trees tire, there is the demand that continues.

Summary of the invention

On the one hand, the invention provides the method for cotyledon Embryo Production softwood tree cotyledon somatic embryo in the past.This aspect method of the present invention comprises the following steps: that (a) is in being enough to the aseptic diluted medium of the volume of the porose material surface of submergence at least, cotyledon embryos before a plurality of are dispensed to level place on the porose material on the pore-free surface, thus cotyledon embryo before scattering equably; (b) from porose material, remove aseptic diluted medium, thereby the preceding cotyledon embryo of uniformly dispersing is intercepted and captured on porose material; And the preceding cotyledon embryo that (c) will intercept and capture on porose material contacts the period that is enough to generate softwood tree cotyledon somatic embryo with the growth substratum.

On the other hand, the invention provides the method for producing softwood tree cotyledon somatic embryo from the softwood tree somatocyte.This aspect method of the present invention comprise the following steps: (a) with the softwood tree somatic cell culture in inducing culture, generate embryogenesis cells; (b) embryogenesis cells of preparation in the step (a) is cultivated in the liquid-retentive substratum cotyledon somatic embryos of coniferous trees tire before forming; (c) in being enough to the aseptic diluted medium of the volume of the porose material surface of submergence at least, the cotyledon embryo level of being assigned to before step (b) preparation a plurality of is placed on the porose material on the pore-free surface, thus cotyledon embryo before scattering equably; (d) from porose material, remove aseptic diluted medium, thereby the preceding cotyledon embryo of uniformly dispersing is intercepted and captured on porose material; And the preceding cotyledon embryo that (e) will intercept and capture on porose material contacts the period that is enough to generate softwood tree cotyledon somatic embryo with the growth substratum.

The cotyledonary embryos tire does not have uniformly dispersing in the reciprocity method of growing on the substratum, and the inventive method produces the somatic embryos of coniferous trees tire of higher yield.In some embodiments,,, cotyledon embryos before a plurality of are distributed in the aseptic diluted medium less than the density of 0.1g wet cell weight with porose material per square inch as porose material 0.005-0.1g wet cell weight per square inch.In some embodiments,,, cotyledon embryos before a plurality of are assigned in the aseptic diluted medium less than the density of 0.05g wet cell weight with porose material per square inch as the density of porose material 0.001-0.05g wet cell weight per square inch.

Method of the present invention if necessary, can be used for for example preparing the somatic embryos of coniferous trees tire, and described somatic embryos of coniferous trees tire can be used for maturing step afterwards and/or can be germinateed to generate growing up to ripe acerose softwood tree plant.Therefore, for example, the inventive method can be used for the acerose clone of productive estabilishment, and this clone has one or more required features, improves as fast growth or lumber quality.For example, the somatic embryos of coniferous trees tire group who utilizes the inventive method to produce can be used for the needle woodlot or the woods that production has one or more required features (improving as fast growth or lumber quality).These trees can be used for producing timber-work again.

The accompanying drawing summary

The aforementioned aspect of the present invention and many advantages that accompanies, by reference detailed description hereinafter, the easier cognition that becomes in conjunction with the accompanying drawings the time becomes better understood simultaneously, wherein:

Fig. 1 has illustrated the exemplary plating frame that comprises the porose material that places on the carriage, is used for the embodiment of the inventive method; With

The photographs that Fig. 2 A-H provides proves, relatively is improved according to the cotyledon embryo's of the inventive method plating growth and dressing plate inoculation method, as described in embodiment 3.Fig. 2 A-D shows the control of drop plating (drop plating) method to genotype A, E, F and B respectively.Fig. 2 E-H shows that respectively liquid dissemination converges the result of the plating method of sprawling (liquiddispersion confluent spread plating method) to genotype A, E, F and B.

DESCRIPTION OF THE PREFERRED

Unless this paper has clearly definition in addition, the implication of all terms that this paper uses is identical with the implication that those skilled in the art of the present invention should understand.

The term that this paper uses " embryonic cell " is meant any cell, comprise can be organized formative tissue or organ, derived from the cell of the plant of Coniferales, when handling with the inventive method, it can produce one or more somatic embryos of coniferous trees tires.Therefore, term " embryogenesis cells " comprises, for example the softwood tree embryonal suspensor mass.

The term that this paper uses " preceding cotyledon embryo " is meant the embryo who does not have any cotyledon as yet.

The term that this paper uses " cotyledon embryo " is meant the embryo with at least one cotyledon.

The inventor finds that the cotyledonary embryos tire does not have uniformly dispersing in the reciprocity method of growing on the substratum before, and method of the present invention produces the somatic embryos of coniferous trees tire of higher yield.The inventor further observes, according to method of the present invention, with porose material per square inch less than the density of 0.1g wet cell weight, as the density of porose material 0.001-0.1g wet cell weight per square inch, cotyledon embryo before the plating, with with high-density plating more and/or utilize the preceding cotyledon embryo of traditional dropper drop method plating to compare, producing per unit area cotyledon embryo yield increases, as embodiment 2 and 3 further as described in.

According to aforementioned content, on the one hand, the invention provides the method for cotyledon embryo generation softwood tree cotyledon somatic embryo in the past.This aspect method of the present invention comprises the following steps: that (a) is in being enough to the aseptic diluted medium of the volume of the porose material surface of submergence at least, cotyledon embryos before a plurality of are dispensed to level place on the porose material on the pore-free surface cotyledon embryo before scattering equably thus; (b) from porose material, remove aseptic diluted medium, thereby the preceding cotyledon embryo of uniformly dispersing is intercepted and captured on porose material; And the preceding cotyledon embryo that (c) will intercept and capture on porose material contacts the period that is enough to generate softwood tree cotyledon somatic embryo with the growth substratum.

Method of the present invention can be used for from any softwood tree, belongs to the member such as pine (Pinus), as torch pine (Loblolly Pine) (Pinus taeda) and pine (Radiata pine), produces the cotyledon somatic embryo.In addition, as embodiment, the inventive method can be produced Pseudotsuga menziesii (Mirbel) Franco cotyledon somatic embryo.

According to the inventive method, will plating a plurality of before cotyledon somatic embryos of coniferous trees tire be suspended in the aseptic diluted medium of certain volume, described volume is enough to submergence at least and places porose material surface on the no pore matrix.Utilize said method, can generate a plurality of preceding cotyledon embryos.For example, the suspension culture of immature somatic embryo (preceding cotyledon embryo) can be cultivated in the liquid-retentive substratum, and allows cell precipitation.Utilize any suitable method then, the method shown in embodiment 2, the volume (SCV) of measurement sedimentation cell.Then the SCV of aequum dilutes with aseptic diluted medium, is used for by the desired density plating.In some embodiments, in order to be enough to the surface of the porose material that submergence at least is connected with the plating frame, the amount that is used to dilute the diluted medium of SCV is selected based on the surface-area of this frame.For example, SCV can use at least about 3-4 times or the more amount dilution of the aseptic diluted medium of SCV volume.

Aseptic diluted medium can be to keep embryo's viability and any suitable liquid nutrient medium of keeping its developmental condition, for example, keeps substratum or exemplary diluted medium shown in the following table 2.

In some embodiments of present method, preceding cotyledon embryo is with the low density plating, supposes the about 0.1g/ml SCV of average weight in wet base as porose material surface covered per square inch less than about 0.1g wet cell weight.The average weight in wet base of SCV can be determined as described in embodiment 2.For example, the embryo can be with porose material plating area per square inch less than 0.05g, or comes plating less than the 0.025g wet cell weight.In some embodiments, preceding cotyledon embryo is with the low density plating to about 0.1g wet cell weight scope (for example porose per square inch material plating area 1ml SCV to 0.01ml SCV) of the about 0.001g of porose material plating area per square inch.In one embodiment, preceding cotyledon embryo is so that the about 0.01g of porose material plating area is to the density plating of about 0.08g wet cell weight scope per square inch, and the about 0.02g of for example porose per square inch material plating area is to about 0.05g wet cell weight.

The pore diameter range that is used to implement porose material of the present invention is from about 5 microns to about 1200 microns, as from about 50 microns to about 500 microns, and as from about 70 to about 150 microns, 100 microns according to appointment.Porose material is planar normally, and can be any required shape and size.The selection of the shape and size of porose material is convenient to operation and is placed as growing on the substratum at growth matrix.Suitable shape comprises square, rectangle or circle.Exemplary size be surface-area from about 14 square inches to 28 square inches or bigger, as 50 square inches, 100 square inches, up to 500 square inches or bigger.Preferred porose material is sterilizable, and has enough intensity to resist tearing when promoting this material, and described lifting is the follow-up phase that is transferred to the somatic embryo production method for the somatic embryo after will being coated with.Useful porose examples of material comprises film, nylon fiber, knitmesh (for example, nylon, stainless steel or plastics) and polymer fiber.In some embodiments, porose material is non-absorption.In some embodiments, porose material is knitmesh, such as stainless steel or nylon wire.

According to the embodiment of the inventive method, porose material such as knitmesh, is used for plating and supports plant tissue in the etap of plant somatocyte embryo production.Before cotyledon somatic embryo initial allocation to level place on the porose material in plane on the pore-free surface.Pore-free surface can be any suitable sterile surfaces, for example, solid or semisolid medium surface, such as containing the petri dish of growing substratum, but or other any aseptic or disinfecting surfaces that can retaining liquids, such as plastics, rubber or glass surface.In some embodiments, pore-free surface is that semisolid contained in the box as the cambro box is grown substratum.In some embodiments, pore-free surface is contained in the bioreactor vessel, but wherein said bioreactor vessel is emptying.

In an embodiment of present method, porose material is connected to the plating frame.The representative example of plating frame 10 is shown in Fig. 1.As shown in Figure 1, plating frame 10 comprises the porose material 20 in plane that links to each other with carriage 30 around porose material 20.Optional handle 40A, the 40B that links to each other with carriage 30 that provide.But the plating frame is preferably made with pasteurization material.For example, carriage 30 can be made by metal or plastic material.But handle 40A, 40B can be made by any suitable pasteurization material, as the autoclavable pipe.The illustrative methods of structure coating frame 10 is described among the embodiment 2.In one embodiment, carriage 30 is metals, and porose material is a nylon wire, and this net linked to each other with above-mentioned frame before the high pressure heating, and appropriateness is shunk after the high pressure heating, produces the plating surface that is connected tension and basic horizontal with frame.

According to the inventive method, in being enough to the aseptic diluted medium of the volume of the porose material surface of submergence at least, cotyledon embryos before a plurality of are dispensed on the porose material that places on the pore-free surface.Thus, preceding cotyledon embryo uniformly dispersing spreads all over the immersed surface of porose material.In some embodiments, mix or stir the embryo of distribution lightly, to promote the distribution of embryo in aseptic diluted medium.Stir gently and can realize by any suitable manner, for example by adopting contact embryo's instrument, or by porose material and/or there is not the vibration of pore matrix.

In case the preceding cotyledon embryo who distributes is dispersed on the porose material substantially equably, then shifts out aseptic diluted medium from porose material.In one embodiment,, shift out aseptic diluted medium, intercept and capture the preceding cotyledon embryo of uniformly dispersing on the porose material surface thus from porose material by porose material is vertically lifted from no pore matrix.For example, the porose material that links to each other with the plating frame 10 that comprises

handle

40A, 40B can adopt any suitable manner to mention by handle is vertical, as manually or pass through robot apparatus.

In an alternative embodiment, in case the basic uniformly dispersing of preceding cotyledon embryo that distributes is on porose material, then be reduced to the below of porose material surface by volume with aseptic diluted medium, and shift out aseptic diluted medium, on porose material surface, intercept and capture the preceding cotyledon embryo of uniformly dispersing thus.Reduce any method that aseptic diluted medium volume can adopt the distribution of avoiding hindering the plating cell, for example suction, emptying, incline to, or blot aseptic diluted medium.

Then, the preceding cotyledon embryo of the uniformly dispersing of intercepting and capturing on porose material surface contacts the period that is enough to produce softwood tree cotyledon somatic embryo with the growth substratum.

In one embodiment, porose material or continue or grow substratum with liquid off and on to contact.For example, porose material can place and be soaked on the absorption pad of growing substratum, and feasible growth substratum is by porose material and contact the embryo.Porose material, such as the nylon wire that carries embryonic cell, the enclosed space of packing into usually, described space contains wetly to guarantee that the embryo keeps moist.In another embodiment, porose material places on the growth matrix that comprises solid or semi-solid growth substratum.

The growth substratum that is used for the inventive method contains the nutrient of keeping somatic embryo.Maltose and glucose can be included in grows in the substratum, main or unique sugared source of using as somatic embryo.Useful maltose and glucose concn scope from about 1% to about 2.5%.Suitable growth substratum does not generally comprise growth promoting hormones, such as plant hormone and phytokinin, but can comprise the hormone dormin.When dormin was used to grow substratum, its concentration range of usually utilizing was about 1mg/L about 200mg/L extremely.Grow substratum and can contain gelling gum (gellangum), concentration is generally up to about 0.40%.Grow the osmolarity of substratum and utilize permeate agent (osmoticant) as PEG 8000 molecular weight, be adjustable to the value that falls in the required scope, 250mM/Kg is to about 450mM/Kg according to appointment.Generally speaking, 300-350mM or higher osmolarity are useful.The example that suitable liquid or solid is grown substratum is provided in embodiment 1 and 2.

As embodiment, preceding cotyledon somatic embryos of coniferous trees tire can be cultivated at porose material, on nylon wire or film, this material at least off and on grow substratum under 10-30 ℃ temperature, as 15-25 ℃, or as under 20-23 ℃, contacted for 4 week-14 weeks, as 8-12 week, or 12 weeks according to appointment.

In one embodiment, before cotyledon somatic embryos of coniferous trees tire cultivate and to grow on the porose material that substratum contacts with liquid, described growth substratum is applied to absorption base, such as the matrix of making by Mierocrystalline cellulose (for example cellulosic fibre), as one or more layers filter paper, or some other absorbing material.Matrix absorption liquid is grown substratum, and this substratum is by placing the porose material on the matrix, and contact places cotyledon somatic embryo before the softwood tree on the porose material.Grow the growth of the preceding cotyledon somatic embryo of substratum promotion softwood tree, form the cotyledon somatic embryo.

In another embodiment, preceding cotyledon somatic embryos of coniferous trees tire is cultivated on the porose material, and described porose material use spraying gun and liquid are grown substratum and contacted, and this spraying gun is with the growth substratum porose material of spraying.Somatic embryo places the upper surface of porose material, and the opposite lower surface of porose material is grown the substratum spraying with liquid.As further embodiment, the porose material of portable object cell stage can place liquid to grow on the substratum, and this growth substratum comprises the rotation stirring rod, and this stirring rod rotation is enough fast, liquid is grown the lower surface that substratum upwards is sprayed to porose material.

On the other hand, the invention provides the method for producing softwood tree cotyledon somatic embryo from the softwood tree somatocyte.This respect method of the present invention comprises the following steps: that (a) cultivates the softwood tree somatocyte in inducing culture, generates embryogenesis cells; (b) embryogenesis cells of preparation in the step (a) is cultivated in the liquid-retentive substratum cotyledon somatic embryos of coniferous trees tire before forming; (c) in being enough to the aseptic diluted medium of the volume of the porose material surface of submergence at least, cotyledon embryo before step (b) preparation a plurality of is dispensed on the porose material that places on the pore-free surface cotyledon embryo before scattering equably thus; (d) remove aseptic diluted medium from porose material, thereby the preceding cotyledon embryo of uniformly dispersing is intercepted and captured on porose material; And the preceding cotyledon embryo that (e) will intercept and capture on porose material contacts the period that is enough to generate softwood tree cotyledon somatic embryo with the growth substratum.

Therefore, in some embodiments, the softwood tree somatic cell culture among the inducing culture or on, generate embryogenesis cells.Embryogenesis cells can produce one or more cotyledon somatic embryos of coniferous trees tires.The example of embryogenesis cells is embryonal suspensor mass (ESMs).

Inducing culture generally includes inorganic salt and organic nutrient material.The osmolarity of inducing culture is generally about 160mM/kg or lower, but also may be up to 170mM/kg.Inducing culture generally includes tethelin.The example that can be included in the hormone in the inducing culture has plant hormone (for example, 2,4 dichloro benzene ethoxyacetic acid (2,4-D)) and phytokinin (for example, 6-benzylaminopurine (BAP)).The usable concentration of plant hormone for example is 1mg/L to 200mg/L.The usable concentration of phytokinin for example is 0.1mg/L to 10mg/L.

Inducing culture can comprise absorbent components, when especially using the tethelin of high level.Absorbent components can be when implementing the inventive method used concentration nontoxic to embryogenesis cells, can absorb any component that produces and be present in the toxic compounds in the substratum in the hormone that promotes growth and the embryo development procedure by vegetable cell.The unrestricted example of useful absorbent components comprises gac, soluble poly (V-Pyrol RC), insoluble poly-(V-Pyrol RC), activated alumina and silica gel.The amount of absorbent components for example is that about 0.1g/L is to about 5g/L.Inducing culture is solid normally, and can import jelling agent and solidify.The example of the inducing culture that the invention process is useful is set forth in embodiment 1.

The softwood tree somatocyte usually among the inducing culture or on, under 10-30 ℃ of temperature, as 15-25 ℃, or as 20-23 ℃, cultivate 3-12 week, as 8-10 week, or 8 weeks according to appointment.

Keeping substratum can be solid medium, perhaps can be liquid nutrient medium, and described liquid nutrient medium can stir to promote the embryo that the growth and the propagation of tissue take place.Keep the osmolarity of the osmolarity of substratum, generally in the 180-400mM/kg scope usually above inducing culture.Keep substratum and can comprise the nutrient of keeping embryo's generation tissue, and can comprise the cell fission of promotion embryo generation tissue and the hormone of growth, such as one or more plant hormones and/or phytokinin.Usually, the concentration of hormone in keeping substratum is lower than its concentration in inducing culture.

Although be not essential, general hope is kept and is comprised in the substratum that maltose is as unique or main metabolizable sugar source.The example of useful maltose concentration scope is about 1% to about 2.5%.The suitable example of keeping substratum is set forth among the embodiment 1 of this paper.

The softwood tree embryogenesis cells usually keep among the substratum or on, under 10-30 ℃ of temperature,, or, cultivated maximum 6 months as 20-23 ℃ as 15-25 ℃, carry out weekly time cultivating.

The softwood tree embryogenesis cells maybe is transferred to the fresh substratum of keeping usually once in a week when growth exhausts nutrient media components.

Useful growth substratum is in preceding description.After or periodicity continuous with the growth substratum contacted cultivation, the cotyledon somatic embryo can be chosen wantonly and be transferred to maturation medium, is transferred to layering substratum (stratification media) then, goes through further incubation period.

The inventive method can be used for, and for example, produces the individual acerose clone with one or more required features (as fast growth).Therefore, in one aspect, the invention provides the identical softwood tree cotyledon somatic embryo group's of production heredity method.This aspect method of the present invention respectively comprises a step: before the softwood tree that heredity is identical the cotyledon somatic embryo with grow substratum continuously or the porose material that periodically contacts (for example, porose nylon wire) goes up the period that cultivation is enough to the softwood tree cotyledon somatic embryo that cotyledon somatic embryo production heredity is identical in the past, wherein grow substratum by porose material and contact cell stage.

Need, the somatic embryos of coniferous trees tire that utilizes the inventive method to produce can be chosen germination wantonly, and formation can grow up to acerose softwood tree plant.The cotyledon embryo also can place in the synthetic seed, is used for germinateing subsequently.Softwood tree cotyledon somatic embryo can, for example, germinate the germination substratum described in the embodiment of the invention 2 on the solid germination substratum.The plant that germinates is transferred to soil, further growth.For example, the plant of germination can be planted in the soil in greenhouse, and allows its growth before migrating to outdoor place.Usually, illumination softwood tree cotyledon somatic embryo stimulates germination.Usually, the institute of the inventive method except that germinateing, carries out in steps all in the dark.

With embryogenesis cells with high-density more and/or in the presence of excess liq plating equivalent processes relatively, the inventive method produces the somatic embryos of coniferous trees tire of the higher yield of every plating surface-area, as further describing at preceding embodiment 2 and 3.

The inventive method can be used for, and for example, production has the individual acerose clone of one or more required features such as fast growth.Method described herein can be used for the identical ripe somatocyte softwood tree embryo group of production heredity.

The following example only illustrates implements the best mode that the present invention thinks at present, should not be interpreted as limiting the present invention.

Embodiment 1

This embodiment shows the exemplary process of the present invention of producing somatocyte pine tree embryo from torch pine.

The megagametophyte that contains zygotic embryo took out from seed in after fertilization 4-5 week.Remove the seed skin, but the embryo does not cut out from gametophyte on every side further except that excision megarchidium end.Cone is stored in 4 ℃, until use.Before removing immature embryo, at once, utilize first washing and washing composition to handle, then with 15%H ₂O ₂Sterilized 10 minutes, and seed is carried out disinfection.Explant is being handled the back with the sterile distilled water thorough washing at every turn.

Table 1 and 2 has been listed the exemplary compositions of producing the useful substratum of pine tree somatic embryo.

Table 1

Torch pine (Pinus Taeda) basic medium (BM)
		Component	Concentration (mg/L)
NH ₄NO ₃	150.0
		KNO ₃	909.9
KH ₂PO ₄	136.1
		Ca(NO ₃) ₂·4H ₂O	236.2
CaCl ₂·4H ₂O	50.0
		MgSO ₄·7H ₂O	246.5
Mg(NO ₃) ₂·6H ₂O	256.5
		MgCl ₂·6H ₂O	50.0
KI	4.15
		H ₃BO ₃	15.5
MnSO ₄·H ₂O	10.5
		ZnSO ₄·7H ₂O	14.4
NaMoO ₄·2H ₂O	0.125
		CuSO ₄·5H ₂O	0.125
CoCl ₂·6H ₂O	0.125
		FeSO ₄·7H ₂O	27.86
Na ₂EDTA	37.36
		Maltose	30,000
Inositol	200
		The acid hydrolysis casein	500
L-glutaminate	1000
		Thiamines-HCl	1.00
Pyridoxol-HCl	0.50
		Nicotinic acid	0.50
Glycine	2.00
		Gelrite ⁺	1600
PH regulator to 5.7

⁺Expression: solid medium then uses if desired

Table 2

The substratum in different treatment stage is formed
		BM ₁-inducing culture	BM+2,3-D (15 μ M)+kinetin (2 μ M)+BAP (2 μ M).
BM ₂-keep substratum	BM+2,3-D (5 μ M)+kinetin (0.5 μ M)+BAP (0.5 μ M).Solid medium then adds GELRITE (1600mg/L) if desired.
		Diluted medium	BM+10mg/mL dormin+100-1000mg/mL adds inositol+2.5% maltose.Add following aminoacid mixture: L-proline(Pro) (100mg/L), altheine (100mg/L), L-arginine (50mg/L), L-L-Ala (20mg/L), and L-Serine (20mg/L).Preferably do not exist and keep hormone.
BM ₃-growth substratum	BM+25mg/L dormin+12%PEG-8000+800mg/L adds inositol+0.1% gac+1% glucose+2.5% maltose.Add following aminoacid mixture: L-proline(Pro) (100mg/L), altheine (100mg/L), L-arginine (50mg/L), L-L-Ala (20mg/L), and L-Serine (20mg/L).When the needs solid medium, add GELRITE (2500mg/L).
		BM ₅-layering substratum	Omit the BM that revises behind dormin and the PEG-8000 ₃When the needs solid medium, add GELRITE (2500mg/L).
BM ₆-germination substratum	Replace the BM that revises after the maltose with 2% sucrose.Inositol is reduced to 100.0mg/L, and glutamine and acid hydrolysis casein are reduced to 0.0mg/L.FeSO ₄7H ₂O is reduced to 13.9mg/L, and Na ₂EDTA is reduced to 18.6mg/L.Add 0.8% agar and 0.25% gac.

Induce: the aseptic gametophyte that will have complete embryo places solid BM ₁On the substratum, and under 22-25 ℃, maintained 3-5 week in the environment with 24 hours half-light cycles.Time span depends on the concrete genotype of being cultivated.When the time finishes, form the white mucus group that links to each other with former explant.Microscopy discloses numerous body early embryos usually and links to each other with group.These generally are characterized by long thin-walled handle and link to each other with the microcephaly who has fine and close kytoplasm and macronucleus.

May be up to 150mM/kg under some situation of the osmolarity of inducing culture.It is about 120mM/kg under the normal conditions, even lower (as 110mM/kg).

Before the keeping and breed of cotyledon embryo: the body early embryo that takes out the group that will generate from inductive phase at first places BM ₂Gelling keep with proliferated culture medium on.This is different from inducing culture and is, tethelin (plant hormone and phytokinin) is lowered at least one complete order of magnitude.The osmolarity of this substratum is 130mM/kg or higher (for torch pine, usually in about 120-150mM/kg scope).Temperature still is 22-25 ℃ in the dark.The embryo is before being transferred to the further inferior cultivation of liquid nutrient medium, at BM ₂Cultivated 12-14 days on the solid medium.The composition of this liquid nutrient medium and BM ₂Identical, but lack jelling agent.Embryo when the solid phase of keeping finishes is similar to those embryos of inductive phase in appearance usually.Cultivate 5-6 after week last time at the liquid-retentive substratum, formed senior body early embryo.These embryos are characterised in that the smooth embryo head that has many handles, estimate generally to have 100 above individual cells.

Fetal development: fetal development is according to following embodiment 2 and 3 described carrying out.

In fact the seepage force of this growth substratum can be increased to and surpass the seepage force of keeping substratum.Have been found that osmolarity up to 300mM/kg in addition higher be favourable.Grow preferably 22-25 ℃, implement under the dark fully, the embryo grows up to cotyledon.General several weeks of development time are as 7-12 week.

Layering: cotyledon embryo plant division (singulated) also is transferred to layering substratum BM ₅This substratum is similar to the growth substratum, but lacks dormin, PEG-8000 and gelling gum.The embryo is cultivating 3-6 week on the layering substratum, between about 1 ℃-Yue 10 ℃, under the dark.

Water conditioning: mention the still ripe embryo on porose material from growth matrix, and place H ₂O relative humidity is in 97% the closed container, to schedule to last about 3 weeks.

Germinate: the ripe embryo of conditioning places solid BM ₆Germinate on the substratum.This substratum is to lack the basic medium of producing hormone, revises by reducing sucrose, inositol and organonitrogen.Under 23-25 ℃ envrionment conditions, with embryo's incubation at BM ₆The enough time has young root, hypocotyl, green cotyledon structure and the epicotyl that reaches full growth up to the seedling that produces on the substratum.

Owing to reduced carbohydrate concentration, the seepage force of germination substratum further reduces, and is lower than to grow under the substratum.Under its common about 150mM/kg (100mM/kg according to appointment).

Embodiment 2

This embodiment describes the structure and the application thereof of exemplary plating frame of the embodiment of the inventive method.

The structure of plating frame: the structure of metal plate inoculation frame is with the bonding 100 micrometer nylon knitmesh of silicone.Plating frame (10) is shown in Fig. 1.In the embodiment of plating frame (10) shown in Figure 1, nylon knitmesh (20) be shaped as rectangle, long 7 feet, wide 4 inches, 28 square inches of exposed surface area.(40A 40B) is connected with metal frame (30) the pipe handle, so that plating frame (10) is mobile.Add silica bead along edge and the middle part of crossing frame, to create 2 plating district (not shown)s of separating that size is identical.Plating frame (10) high pressure heating then because nylon knitmesh (20) is contracted on the metal frame (30), produces the plating surface of tension in high pressure heat-processed.

At plating frame upper flat plate inoculating cell

Plating is with the preparation of SCV: allow the somatic embryos of coniferous trees fetus cells precipitation of long genotype A in the propagating culture medium (preparation as shown in table 4) of 1 liter of Ehrlemeyer flask.Measure sedimentary cell volume (SCV) by line on flask, the supernatant liquor on the sedimentation cell is removed with suction through the agglomerating glass stick.Then, sedimentary cell is resuspended in the aseptic diluted medium of pressing embodiment 1 described preparation of 3 * SCV magnitude of recruitment.

Measure the weight in wet base of SCV:, measure SCV as mentioned above in order to measure the weight in wet base of SCV.Then, utilize Buchnar funnel (13 inches high) to remove supernatant liquor, 1ml SCV sample is placed on wetting in advance VWR level 417 filter paper of weighing in advance.(30 seconds) carry out check weighing on 4-point balance at a fixed time.The genotypic weight in wet base measurement of 4 kinds of representativenesses of every ml SCV is shown in the following table 3.

Table 3

Genotype	Weight in wet base mg/ml SCV
		A	92mg/ml
B	102mg/ml
		C	102mg/ml
D	118mg/ml

From the result shown in the table 3 as seen, 4 of test kinds of genotypic average weight in wet bases are 103.5mg/ml SCV.Therefore, the average weight in wet base of 4 of test kinds of genotypic 1ml SCV is about 0.1mg/mL SCV.

The plating frame (10) of preparation as mentioned above is provided, and places semi-solid surface of growing substratum (preparation as shown in table 5).Measure the volume of the sedimentation cell of rinsing then, and with density (0.3g/14 square inch=0.02g/ square inch) plating of 3ml SCV forebody to the plating frame, and latter half of with density (1.2g/14 square inch=0.08g/ square inch) plating of 12ml SCV to the plating frame, cumulative volume separately is respectively 9ml and 36ml.

Carry out the cell plating on the plating frame, described plating frame places the contained semisolid of box to grow on the surface of substratum.This makes cell and substratum intersperse among nylon wire equably, is intended to make the minimize variations of the cell density and the initial cell degree of depth.In case the cell of plating is dispersed on the buried nylon wire equably, plating frame (10) is vertically lifted from the semi-solid substratum of growing, intercept and capture the embryo of uniformly dispersing thus, and make the excessive substratum of staying in first box scatter.In new box, the plating frame that will contain the embryo of uniformly dispersing places the semisolid identical with first box prescription to grow on the surface of substratum then.Then, allow the plating cell on the plating frame grow for 12 weeks, and monitoring total biomass, embryonal suspensor mass and embryonic structure form, as shown in table 6 below.

Table 4

Breed/keep substratum (torch pine)
		Composition	Concentration (mg/L)
NH ₄NO ₃	150.0
		KNO ₃	909.9
KH ₂PO ₄	136.0
		Ca(NO ₃) ₂·4H ₂O	236.15
CaCl ₂·2H ₂O	50.0
		MgSO ₄·7H ₂O	246.5
Mg(NO ₃) ₂·6H ₂O	256.5
		MgCl ₂·6H ₂O	50.0
KI	4.15
		H ₃BO ₃	15.5
MnSO ₄·H ₂O	10.5
		ZnSO ₄·7H ₂O	14.4
NaMoO ₄·2H ₂O	0.125
		CuSO ₄·5H ₂O	0.125
CoCl ₂·6H ₂O	0.125
		FeSO ₄·7H ₂O	27.86
Na ₂EDTA	37.36
		Maltose	30,000
Inositol	200
		The acid hydrolysis casein	500
L-glutaminate	1000

Thiamines-HCl	1.00
		Pyridoxol-HCl	0.50
Nicotinic acid	0.50
		Glycine	2.00
^*Gelrite ⁺	1600 ^*
		2，4D(10mg/mL)	1.1mg/L
6-BAP(10mg/mL)	0.1mg/L
		Kinetin (10mg/mL)	0.1mg/L
^*ABA(2mg/mL)	1.0mg/L ^*
		PH regulator to 5.7	( ^*=optional)

Table 5

Grow substratum (torch pine)
		Composition	Concentration (mg/L)
NH ₄NO ₃	150.0
		KNO ₃	909.9
KH ₂PO ₄	136.0
		Ca(NO ₃) ₂·4H ₂O	236.15
CaCl ₂·2H ₂O	50.0
		MgSO ₄·7H ₂O	246.5
Mg(NO ₃) ₂·6H ₂O	256.5
		MgCl ₂·6H ₂O	50.0
KI	4.15
		H ₃BO ₃	15.5
MnSO ₄·H ₂O	10.5
		ZnSO ₄·7H ₂O	14.4
NaMoO ₄·2H ₂O	0.125
		CuSO ₄·5H ₂O	0.125
CoCl ₂·6H ₂O	0.125

FeSO ₄·7H ₂O	27.86
		Na ₂EDTA·2H ₂O	37.36
Maltose	25,000
		Glucose	10,000
Inositol	100-1000
		The acid hydrolysis casein	500
L-glutaminate	1000
		Thiamines-HCl	1.00
Pyridoxol-HCl	0.50
		Nicotinic acid	0.50
Glycine	2.00
		Proline(Pro)	100
The L-arginine	50
		Altheine	100
The L-L-Ala	20
		The L-Serine	20
PEG	100000
		Gac	1000
Gelrite ⁺	2500
		ABA(2mg/mL)	25.0mg/L
PH regulator to 5.7

The result:

Cultivate after 12 weeks, check in the plating frame that total biomass, embryonal suspensor mass (ESM) and the embryonic structure with the culture of different densities plating forms.Selected embryo is meant at least 4 cotyledons of existence and does not have the incompleteness of vast scale.The results are shown in the following table 6.

Table 6

Plating density	Eventually wet biomass (g)	Total dry biomass (mg)	Do DSM (mg)	Dried embryo (mg)	Selected embryo number
						The SCV of low density (half frame) 3ml plating	Be total to 17.0g (SCV of 5.67 g/ml platings)	Totally 1277.6 mg (SCV of 425.87 mg/ml platings)	Be total to 235mg (SCV of 78.33mg/ml plating)	Be total to 291.8mg (SCV of 97.27mg/ml plating)	Totally 720 (SCV of 240/ml plating)
The SCV of high-density (half frame) 12ml plating	Be total to 14.0g (SCV of 1.17 g/ml platings)	Totally 1302.4 mg (SCV of 108.53 mg/ml platings)	Be total to 138.7mg (SCV of 11.56mg/ml plating)	Totally 312.0 (SCV of 26.0 mg/ml platings)	Totally 771 (SCV of 64.25/ml plating)

As above shown in the table 6, when the embryo with (for example than low density, 3ml SCV/14 inch (about 0.3g/14 square inch=0.02g/ square inch)) during plating, the propagation subsequently of embryonal suspensor mass and total biomass with the cell output as much of higher density 12ml SCV/14 square inch (about 1.2g/14 square inch=0.08g/ square inch) plating.Shown in table 6 is further, almost identical between low density and high-density with embryo's total amount that every planimeter was produced.Shown in dry weight difference, by improving the growth of embryonal suspensor mass (ESM) greatly, this might realize that for example, low-density dried ESM is 235mg, and the dried ESM of the frame of high-density plating has only 138.7mg.Therefore, (64.25 embryos/mlSCV) compare, the SCV of selected embryo's formation (purpose end product)/ml plating is obviously better in low density plating (240 embryos/ml SCV) with the high-density plating.

Embodiment 3

This embodiment describes, and utilizes the torch pine of four kinds of different genotype, and the liquid dissemination by alibrated pipette drop plating method and embodiment of the present invention converges the plating method of sprawling, and carries out the comparison between embryo's yield.

Method: directly relatively utilize four kinds of different genotype torch pine A, B, E and F of identical biomass, change the plating density of plating SCV simultaneously, carry out direct comparative experiments in the method for plating frame (10) upper flat plate inoculating cell.Measure embryo's yield and germination result.

The embryoid body cell (ESM) of genotype A, B, E and F is grown in the proliferated culture medium (as preparation as described in the embodiment 2) of 1 liter of Erlenmeyer flask.As described in embodiment 2, make the ESM cell precipitation, and measure sedimentary cell volume (SCV).

For each genotype, as the contrast of representing standard drop plating method, 6mlSCV is made 12 (every 0.5ml SCV), direct flat plate be seeded to whole plating frame (7 " * 4 " total area=28 square inch), this frame places the semisolid of shallow plating box to grow on the substratum.In this embodiment, utilize cambro box that two plating frames (10) are housed (Cambro Manufacturing Co., Huntington Beach, CA).In order to test the plating method of sprawling, each genotypic second equal portions 6ml SCV grows the substratum rinsing with 3 * (18ml).Then, 24ml SCV adds that the rinsing culture medium flat plate is seeded on the whole plating frame (28 square inches), described frame places the first semi-solid substratum top of growing of cambro box, causes the ESM cell to swim in the immersed surface that substratum also is dispersed in the plating frame equably.Stir the plating frame gently and help uniformly dispersing.Then, utilize connection handle to keep the plating surface simultaneously in the horizontal direction, the plating frame is vertically lifted from first semisolid medium in the cambro box, intercept and capture the ESM cell of uniformly dispersing thus, make substratum flow through porose net simultaneously.

Then, the plating that will contain the plating cell is frameed shift to the fresh cambro box that contains semi-solid growth substratum (as described in embodiment 2), and grows for 12 weeks.12 weeks, the embryo stood to grow late period to handle to induce germination when finishing, counting, and sample germinates.The normal germination kept the score to there being the white root of 1mm, has the epicotyl blade of about 5 about 5mm of length, do not have the hypocotyl of vast scale to break, and do not have crooked hypocotyl greater than 90 degree.

After 12 growth perioies in week, the total recovery of embryonic development is counted.The results are shown in following table 7.Growth period takes pictures when finishing, as shown in Figure 2.Fig. 2 A-D shows genotype A respectively, E, the contrast of F and B.Fig. 2 E-H shows genotype A respectively, E, and the liquid dissemination of F and B converges the result who sprawls the plating method.

12 weeks utilized layering substratum (described in the embodiment 1) with 4 weeks of embryo's layering when finishing.After 4 weeks of layering, embryo's spraying separates, and water was nursed one's health 10 days.

For each genotype, select 25 embryos to be used for germinateing and estimate.The embryo who selects for germinateing is based on having at least 4 cotyledons and not having overall fault or the hypocotyl cracking.

The result:

Table 7

Genotype	The every box embryo of sessile drop method yield (germination %)	The every box embryo of the method for sprawling yield (germination %)	The multiple that method embryo yield increases is sprawled in utilization
				A	2241(52％)	5416(49％)	2.4
E	2267(15％)	3187(27％)	1.4
				F	1803(8％)	2545(3％)	1.4
B	2356(10％)	3188(19％)	1.3

As shown in table 7,4 kinds of genotype of all tests show, compare with conventional suction pipe drop plate streak, and low density is converged at least 1.3 times of embryo's yield increases of sprawling plate streak or higher.The result of germination studies shows, do not have statistical significant difference utilizing sessile drop method and sprawl between the sample germination success ratio of method plating.

When 4 kinds of genotypic result combinations shown in the table 7, the embryo's overall average control value that utilizes sessile drop method to produce is 2166.7, and embryo's population mean of utilizing liquid dissemination to converge the spreading methods generation is 3692, and embryo's yield is high 1.7 times, and the p value is 0.0708.This is to improve very significantly in embryo's yield, make the genotype plating in single cambro unit, to realize the average yield target of germination yield, and will operate the required unit number of single clone is reduced to and has only one, by contrast, utilize conventional suction pipe drop plate streak to need at least 30 independent unit (culture dish).

The combined result of 4 kinds of genotype records of test is provided in the following table 8.

Table 8

Parameter	Contrast drop plating (6ml SCV, 0.5ml/ drip * 12)	Sprawl plating (6ml SCV is diluted among the 18ml)	The p-value
				Total embryo's yield/box	2167	3692	0.0708
Average embryo yield/ml SCV	180.6	307.6
				The I class is germinateed	17.2	20.9	0.4261
Bud/ml SCV	40	88.9	0.1768
				Average root long (mm)	32.1	23.7	0.1481

Data presentation shown in the last table 8, though long some the difference sign of root, significance,statistical on embryo's yield (the 0.10p value is ended) increases by 70%, and percentage of germination does not have significantly sacrificing not have.Though be not wishing to be bound by theory, the difference of observing the root size may be since with the bigger relevant nutrition problem of increment.The solution of nutrition problem can be by moving to fresh growth matrix with the embryo of plating after for some time, or form to bigger concentration by regulating substratum, and/or regulate plating density based on the growth rate of concrete clone.

As shown in table 8, the plate streak of sprawling of the present invention is compared with the drop plate streak, causes the yield 120% of bud, and prompting is based on every housing unit and surface-area, aspect the embryo's generation and the two the measured cultivation productivity of germinateing, very large improvement is arranged.The sessile drop method of growing in the culture dish is opposite, and observed this improvement allows to scale up to the size on plating surface without limits in embryo's yield.This scaling up improved embryo's amount that every plating unit forms, and reduces the per unit cost by reducing required operational ton.

Though having described, this embodiment utilize the Cambro box as the plating container, but the plating container that is used to grow step after plating can be any suitable plating surface, such as any box type container, such as using commercially available food products preparation container, this container is heat-staple, and has lid.

Liquid dissemination converges sprawls the successful implementation like this of plating method: with the plating frame on the multiple atresia sterile surfaces that is placed on except that semisolid medium, described surface ratio such as aseptic plastic lid or place silicon chip or rubber pad in can the container of retaining liquid then contacts the plating embryo with the growth substratum.

Liquid dissemination converges sprawls the successful implementation like this of plating method: at first bed board is enough to the aseptic diluted medium of the amount on submergence plating surface, and described surface comprises the porose material that places on solid (atresia) matrix.Then, volume required SCV is added into diluted medium, and mixes gently with suction pipe.Then optional by means of suction pipe and/or stir the first plating surface gently, make floating cell scatter.Pick up the plating frame, and place and comprise on second growth matrix of growing substratum, and incubation as mentioned above.

At last, utilize plating, converge spreading methods and also be successfully used to generate the embryo from multiple torch pine genotype to placing semi-solid equal portions of growing the 1-12ml SCV on the whole plating frame on the substratum (7 " * 4 ").

Although the preferred embodiment of the invention is illustrated and describes, be appreciated that under the situation that does not deviate from essence of the present invention and category and can carry out various variations.

Claims

1. produce the method for softwood tree cotyledon somatic embryo, described method comprises:

A) by cotyledon embryo before a plurality of softwood tree being suspended in the aseptic diluted medium that is enough to the volume of the porose material surface of submergence at least, and cotyledon embryo before the softwood tree in the aseptic diluted medium is dispensed on the porose material, cotyledon embryo before a plurality of softwood tree is uniformly distributed on the porose material with the density less than the 0.1g wet cell weight of porose material per square inch, wherein the pourous wood material package contains the mean pore size scope in 5 microns to 1200 microns hole, and wherein porose material horizontal places on the pore-free surface;

B) remove aseptic diluted medium from porose material, thereby on porose material, intercept and capture cotyledon embryo before the softwood tree of uniformly dispersing; And

C) will intercept and capture before the softwood tree on porose material the cotyledon embryo and grow substratum and contact to the time that is enough to generate softwood tree cotyledon somatic embryo.

2. the process of claim 1 wherein cotyledon embryo before a plurality of softwood tree with porose material per square inch less than the density distribution of 0.05g wet cell weight in the aseptic diluted medium of step (a).

3. the process of claim 1 wherein cotyledon embryo before a plurality of softwood tree with the density distribution of porose material 0.1g to 0.001g wet cell weight per square inch in the aseptic diluted medium of step (a).

4. the process of claim 1 wherein that porose material right and wrong are absorbefacient.

5. the method for claim 4, wherein porose material is knitmesh.

6. the process of claim 1 wherein that porose material is connected with the carriage that handle is arranged.

7. the method for claim 6 is wherein by vertically lifting from carriage no pore matrix and remove aseptic diluted medium from the porose material of step (b).

8. the process of claim 1 wherein by aseptic diluted medium being reduced to the level under the porose material surface, and remove aseptic diluted medium from the porose material of step (b).

9. the process of claim 1 wherein that the growth substratum of step (c) is a liquid nutrient medium.

10. the process of claim 1 wherein that the growth substratum of step (c) is a solid medium.

11. produce the method for softwood tree cotyledon somatic embryo, comprising:

(a) with the softwood tree somatic cell culture in inducing culture, generate the softwood tree embryogenesis cells;

(b) the conifer embryogenic fetal hair of preparation in the step (a) is given birth to cell cultures in the liquid-retentive substratum, form the preceding cotyledon somatic embryo of softwood tree;

(c) by cotyledon somatic embryo before a plurality of softwood tree being suspended in the aseptic diluted medium that is enough to the volume of the porose material surface of submergence at least, and cotyledon somatic embryo before the softwood tree in the aseptic diluted medium is dispensed on the porose material, cotyledon somatic embryo before a plurality of softwood tree of step (b) preparation is uniformly distributed on the porose material with the density less than the 0.1g wet cell weight of porose material per square inch, wherein the pourous wood material package contains the mean pore size scope in 5 microns to 1200 microns hole, and wherein porose material horizontal places on the pore-free surface;

(d) remove aseptic diluted medium from porose material, thereby the softwood tree proter cell leaf embryo of uniformly dispersing is intercepted and captured on porose material; And

(e) will intercept and capture before the softwood tree on porose material the cotyledon somatic embryo and grow substratum and contact to the time that is enough to generate softwood tree cotyledon somatic embryo.