WO1995005064A1 - Procede d'orientation d'embryons vegetaux - Google Patents

Procede d'orientation d'embryons vegetaux Download PDF

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Publication number
WO1995005064A1
WO1995005064A1 PCT/US1993/007804 US9307804W WO9505064A1 WO 1995005064 A1 WO1995005064 A1 WO 1995005064A1 US 9307804 W US9307804 W US 9307804W WO 9505064 A1 WO9505064 A1 WO 9505064A1
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WO
WIPO (PCT)
Prior art keywords
embryos
medium
flotation
flotation medium
oriented
Prior art date
Application number
PCT/US1993/007804
Other languages
English (en)
Inventor
Curtis A. Bryan
William C. Carlson
Michael K. Mckinnis
Original Assignee
Weyerhaeuser Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US07/865,390 priority Critical patent/US5284765A/en
Priority claimed from US07/865,390 external-priority patent/US5284765A/en
Application filed by Weyerhaeuser Company filed Critical Weyerhaeuser Company
Priority to BR9307878A priority patent/BR9307878A/pt
Priority to AU50814/93A priority patent/AU674888B2/en
Priority to PCT/US1993/007804 priority patent/WO1995005064A1/fr
Priority to CA002167501A priority patent/CA2167501C/fr
Priority to NZ255823A priority patent/NZ255823A/en
Publication of WO1995005064A1 publication Critical patent/WO1995005064A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor

Definitions

  • the present invention is directed to a method of ordering plant embryos so that a specified end is always oriented in a given direction.
  • the invention is also concerned with a method of separating viable from nonviable conifer somatic embryos and properly orienting the viable embryos for insertion into artificial seeds.
  • Reliable methods have now been developed for large scale production of plant somatic embryos of a larger number of species using the techniques of tissue culture. In recent years much effort has been directed to development of techniques for embryogenesis of important conifer species.
  • U.S. Patents 4,957,866, 5,034,326, and 5,036,007 are exemplary of such methods.
  • Embryos In culture the embryos are developed to a stage similar to the natural zygotic embryos occurring in mature seeds. For conifers these are very small, those for the species noted above falling in a range of about 2-4 mm in length. Embryos have a bipolar form which anticipates the ultimate plant. One end has a latent radicle or root and the other a whorl of tiny structures that will become the cotyledons of the germinated seed. The cotyledons, or seed leaves, are the first chlorophyll bearing organs and, in a natural seed, take over from the endosperm the ob of providing nutrients to the newly germinated plant. The latent cotyledons look somewhat like a tiny crown situated at one end of the embryos.
  • somatic embryos lack the endosperm of the natural seed, some other means must be found to provide nutrients to the embryo at the time of germination.
  • the embryos may be placed on a solid germination medium containing the necessary carbohydrate and other nutrients, as described in any of the above-noted patents. They may also be placed on a growing medium, or synthetic soil, which is saturated with an appropriate nutrient solution. In both of these cases sterility must be maintained until after the resulting plantlet is well established.
  • a preferred method of germinating a somatic embryo is to incorporate it into an artificial seed containing the essential nutrients. In essence, the artificial seed replaces the seed coat and endosperm of the natural seed.
  • This method has the advantage that the embryos can be outplanted into a nursery bed in essentially the same manner as a natural seed.
  • a number of versions of artificial seeds have been described in the patent literature of the past half decade. Examples are U.S. Patents 4,562,663; 4,583,320; 4,615,141; 4,715,143; 4,777,762; 4,779,376; and 4,780,987 and Canadian Patent 1 ,241 ,552. Most of these are variations on the theme of completely encapsulating a somatic embryo within a hydrophilic gel.
  • the gel may be based on a material such as sodium alginate which can then be insolubilized on only the surface or throughout by cation exchange with a material such as calcium chloride. By insolubilizing at least the surface the resulting artificial seed can be more readily handled.
  • the preferred form of artificial seed described in the above-noted patent application is far more sophisticated in construction than those using simple gel droplets. It requires insertion of the embryo, cotyledon end first, into a thin moisture pervious tube surrounded by a nutrient gel enclosed within an outer capsule which provides the mechanical equivalent of a seed coat.
  • the tube into which the embryo is inserted is barely larger in diameter than the embryo itself. If an economical production rate is to be obtained this process must be mechanized as much as possible.
  • the present invention is directed to a method of delivering viable embryos, oriented cotyledon end first, to a seed assembly point. No additional sorting or orientation is required. From the delivery location the embryos may be handled by robotic or other means for insertion into the inner tube of an artificial seed of the type noted above.
  • the present invention is concerned with the orientation of plant embryos so that a specified end is essentially always oriented in the same direction at a selected location space. It is particularly directed to the orientation of conifer somatic embryos for placement in artificial seeds.
  • the separation of plant embryos according to development maturity has already been noted.
  • the method described uses a stratified aqueous medium having defined density gradients. Not surprisingly, germination success was greater when more mature embryos were selected.
  • the embryos are first introduced into an aqueous flotation medium held in a containment vessel.
  • This flotation medium is of necessity a nonphytotoxic solution of a density slightly higher than that of the embryos to be selected.
  • By precise control of flotation medium density it is possible to separate the embryos into essentially viable and nonviable groups.
  • the viable embryos will float and the nonviable embryos will sink.
  • viable is meant embryos that have a high likelihood of germinating into normal plantlets.
  • the flotation medium is of essentially uniform density throughout. A density gradient within the medium is not generally desirable. It is essential that turbulence within the flotation medium should be minimized and any flow should be of the laminar type.
  • the embryos are retained in the flotation medium for a sufficient time to permit the viable embryos to float when the lower region of the medium, when they are introduced, to an upper region where they can be conveniently removed.
  • the embryos After reaching the upper portion of the flotation medium, the embryos are captured in a flowing fluid transport medium of increased velocity in a manner so that they are swept cotyledon end first into a small diameter conduit.
  • the conduit diameter is preferably of sufficient diameter so that the embryos can be transported without undue friction but small enough so that the embryos cannot turn end-for-end. This helps to maintain the cotyledon end forward orientation.
  • the embryos may be transported for some distance in the conduit to a delivery point. Here they are separated from the transporting fluid in a manner so that the cotyledon end first orientation is preserved. From this point they may be handled by robotic or other means for insertion into artificial seeds or placement on some type of germination substrate.
  • the containment vessel for the flotation medium is of gradually decreasing cross-sectional area from bottom to top. This will increase the concentration of embryos per unit volume of solution as they approach the top of the containment vessel and facilitate removal.
  • the containment vessel is most preferably of conical configuration although other forms are acceptable.
  • a preferred method of introduction of the embryos into the containment vessel is in suspension in an aqueous medium of very similar or identical composition and density to that of the flotation medium. This may be done in either a batchwise or continuous fashion. In the later case the suspended embryos must be introduced in such a manner that no appreciable turbulence is crated in the flotation zone. Those embryos that sink; i.e., those that presumably are nonviable, may be withdrawn from the bottom of the containment vessel in either a continuous or batchwise fashion. In the case of continuous embryo introduction, the flow velocity in the upper part of the vessel will increase as a result of the decreasing cross-sectional area. This velocity increase should not be so great as to cause turbulent flow.
  • the floating embryos may conveniently be captured from the upper region of the flotation medium by introducing a moving stream of fluid transporting medium adjacent the top of the containment vessel. From here they are swept into the transport conduit without loss of orientation.
  • the moving fluid stream may either be introduced around the periphery in some symmetrical manner or it may be introduced axially. In either case it should be directed upwardly; i.e., in the direction of travel of the rising embryos.
  • the transporting medium will also be of similar or identical composition and density to the flotation medium in the containment vessel. This enables this fluid to be recycled as embryo introduction medium or transporting medium or otherwise reused as long as it can be protected from entry of pathogens.
  • the embryos are separated from the transport fluid for further processing. This can be done in a number of ways which are not the subject of the present invention.
  • One such way is by catching the embryos on a moving belt mechanism which allows the transport fluid to drain from the embryos.
  • Increased discrimination between viable and nonviable embryos can be achieved by first desiccating the embryos prior to flotation. Desiccation to no more than about 50% moisture content is very helpful. Preferably the embryos are desiccated about 15% moisture content or even somewhat less. Desiccation should proceed fairly rapidly to facilitate separation of the immature embryos from those that are sufficiently mature. Rapid desiccation stresses the immature embryos so that they generally become nonviable. This can be done by allowing the embryos to come to equilibrium moisture content when held from some time in atmospheres of constant relative humidity at moderate temperatures.
  • the method is most advantageously used with somatic embryos achieved by the techniques of plant tissue culture. It is particularly effective with somatic embryos of plants in the order Coniferales. This includes most of the important softwood species of both the northern and southern hemispheres. It is an object of the present invention to provide a method for ordering plant embryos so that a specified end is essentially always oriented in a given direction.
  • FIGS. 1 and 4 are elevation views, partially in section, of configurations of flotation containment vessels showing alternative embryo removal arrangements.
  • FIGS. 2 and 3 show details of venturi-like embryo removal systems using peripherally introduced fluid medium.
  • FIG. 5 is an overall diagrammatic view of the embryo orientation apparatus.
  • FIG. 6 is a sectional view of one arrangement for separation of oriented embryos from their transporting fluid.
  • FIG. 7 is a sectional view of preferred configuration of an artificial seed.
  • FIG. 1 shows a preferred conical configuration of a containment vessel 2 containing an aqueous flotation medium 4.
  • a side nipple 6 serves as a point for introduction of plant embryos suspended in a fluid medium 7.
  • the introduction medium 7 is preferably of the same composition and at about the same temperature as the flotation medium 4. While a conical configuration is generally preferred for the flotation containment vessel, other configurations such as an elongated vessel with sloping sides are quite suitable.
  • the embryos By making the upper portion of the vessel of lesser cross- sectional area than the lower portion, the embryos, which are now properly oriented, are concentrated and more readily removed.
  • the embryos are introduced in a random configuration into the flotation containment vessel.
  • Flotation medium 4 is adjusted in density sot hat it is barely higher than the density of viable embryos.
  • All fluid media which contacts the embryos must be of a benign, nonphytotoxic nature.
  • All of the media may advantageously comprise a sterile sucrose solution, most usually in the range of 15%-25% concentration. This corresponds to a density at room temperature of about 1.059-1.104 g/cm 3 .
  • the concentration will preferably be in the range of about 18%-22% sucrose.
  • the corresponding density range will be 1.072-1.090 g/cm 3 .
  • Many other materials beside sucrose can be used for formulating the flotation medium; e.g., other metabolizable or nonmetabolizable sugars with or without other added nutrient materials, sorbitol, xylitol, etc.
  • the flotation medium must be essentially nonturbulent above and below the point of embryo introduction. This is of critical importance if the embryos are to assume their natural orientation of cotyledon end upward. While the embryos may be introduced in either batchwise or continuous fashion, this must be done without introducing significant turbulence into the flotation zone.
  • an embryo transport fluid 12 is introduced through a plurality of manifolded peripheral inlets 14 having upturned nozzles 16. This sweeps the now oriented embryos 20 cotyledon end first into a conduit 18 where they are moved by fluid transport to a separation point.
  • Conduit 18 should be of sufficiently small diameter, or the flow rate should be sufficiently great, so that the embryos cannot turn end-for- end or otherwise lose their preferred orientation while being transported to the delivery point.
  • FIG. 3 An alternative manifolded peripheral arrangement for transport medium introduction is shown in FIG. 3.
  • a modified flotation containment vessel 22 has a plurality of inlets 24. These have upturned nozzles 26 formed in the wall of the containment vessel. In order to reduce uneven flow, the inlets should be arranged symmetrically around the neck portion of the vessel 2. Three such inlets have been found to be sufficient in most cases.
  • FIG. 4 A further alternative arrangement for introduction of transport medium is shown in FIG. 4.
  • a modified flotation containment vessel 32 is provided with an axially located tube 34 which serves to introduce the transport medium 12 near the top of the vessel.
  • FIG. 5 shows the overall arrangement of one system that has proved to be very satisfactory.
  • the embryos are suspended in the introduction medium 7 in a container 42. These are allowed to flow by gravity or otherwise introduced into the flotation containment vessel 2 through nipple 6. Immediately after introduction all of the embryos may initially sink. However, shortly thereafter the viable embryos will begin their rise to the upper zone of the flotation medium. After flotation, the selected embryos, now oriented cotyledon end upward, are removed through conduit 18 to a separation mechanism shown generally at 44. The fluid transport medium after separation of the embryos is recycled through tubing 46 and pump 50.
  • Pump 50 may be any suitable low head, low volume pump, such as a peristaltic pump. Conditions must be such that sterility of the transporting medium is maintained if it is recycled. Any overflow fluid can be redirected to vessel 42 containing the unsorted, unoriented embryos.
  • FIG. 6 One form of suitable embryo separation mechanism 44 is seen in FIG. 6.
  • the terminal end of conduit 18 enters a separation head 62, seen here in cross section.
  • a moving porous belt pair having a narrow lower belt 68 and narrow upper belt 70 are fed in opposing relationship through separation head 62.
  • Each of the belts is supported by driven pulleys 72 and idling pulleys 74.
  • a screened orifice 66 leads to a side nipple 64 where transport fluid medium 12 is drawn off for recycle.
  • the freed embryos 76 now still cotyledon end first and generally singulated in end-to-end fashion, are discharged from between the belt pairs 68, 70 where they may be handled by robotic or other means for further processing.
  • a preferred type of artificial seed 80 for conifer somatic embryos is shown in FIG. 7.
  • An outer covering 82 analogous to a protective natural seed coat, is closed at one end and open at the other. This is preferably made of a biodegradable product, such as water resistant paper, but may also be of a thin walled plastic material.
  • a porous inner tube 84 positioned axially within the outer covering 82, holds the embryo. Inner tube 84 is also closed at the end. This may be made of paper but is more preferably formed of a very thin walled plaster-of-Paris casting.
  • the somatic embryo 76 is inserted cotyledon end first into the inner tube.
  • the bulk of the inner portion of the artificial seed is filled with a nutrient gel 86 which serves as an artificial gametophyte for the germinating embryo.
  • Inner tube 84 is sided so that the cotyledon end of the embryo 76 is in contact with the porous inner tube walls in order to absorb nutrients from the gel.
  • the open end of the outer shell 82 is sealed with a wax plug 88. This does not cover the end of inner tube 84, however. After the embryo 76 has been inserted the artificial seed may be further sealed by a thin wax layer 90 to prevent moisture loss and preserve sterility.
  • EXAMPLE Douglas-fir embryos were desiccated by placing them over a saturated solution of Ca(NO 3 ) 2 .4H 2 O for 5-7 days during which time they came to an equilibrium moisture content below 15%. Relative humidity over this solution is about 54%. Prior to desiccation the embryos were hand selected into categories regarded as mature and immature. In a four times replicated experiment a mixture of 15 embryos believed to be mature and 15 believed to be immature were introduced batchwise into a 1 L Erlenmeyer flask configured as shown in FIG. 1. The flotation medium and the medium in which the embryos were suspended when introduced was a 20% sucrose solution have a density of 1.081 g/cm 3 .
  • the transport medium also a 20% sucrose solution, was turned on into the peripheral jets at the top of the flask and the embryos were carried cotyledon end first into the delivery conduit which was made of flexible tubing 2.0 mm in inside diameter.
  • the delivery conduit which was made of flexible tubing 2.0 mm in inside diameter.
  • 163, or 98% of the accepted embryos were delivered cotyledon end first.
  • Germination tests, using an agar gelled germination medium showed that nearly all of the accepted embryos germinated into apparently normal plantlets. Only a very small number of the rejected embryos germinated and, of these, most did not form normal plantlets.

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  • Life Sciences & Earth Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne la disposition d'embryons végétaux qui se caractérise en ce qu'une extrémité désignée de l'embryon soit toujours orientée dans un sens prédéterminé. Les embryons sont de préférence d'abord assez rapidement déshydratés jusqu'à ne présenter qu'une teneur en humidité de 15 % environ. Ils sont ensuite placés en suspension dans un liquide de flottation bénin ayant une masse volumique comprise entre environ 1059 et 1104 g/cm3. La masse volumique doit être ajustée de manière empirique de sorte qu'un nombre prédominant d'embryons viables flottent et d'embryons non viables se déposent au fond. Dans le cas au moins d'embryons somatiques de conifères, ceux-ci flottent, leur extrémité portant les cotylédons latents étant dirigée vers le haut. Après un temps de séparation suffisant dans le liquide de flottation, les embryons orientés sont balayés par un courant liquide et entraînés dans un conduit. Ils y entrent, les extrémités à cotylédons en premier, puis ils sont ensuite transportés vers un point d'évacuation sans perdre cette orientation, et ils sont séparés du liquide porteur. Les embryons, avec les extrémités à cotylédons toujours positionnées en avant, peuvent être ensuite récupérés par un robot ou autre dispositif afin de subir ensuite un traitement, tel que leur insertion dans une graine artificielle.
PCT/US1993/007804 1992-04-08 1993-08-18 Procede d'orientation d'embryons vegetaux WO1995005064A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US07/865,390 US5284765A (en) 1992-04-08 1992-04-08 Method of directionally orienting plant embryos
BR9307878A BR9307878A (pt) 1992-04-08 1993-08-18 Processo para orientar direcionalmente uma multiplicidade dem embrioes de plantas
AU50814/93A AU674888B2 (en) 1992-04-08 1993-08-18 Method of directionally orienting plant embryos
PCT/US1993/007804 WO1995005064A1 (fr) 1992-04-08 1993-08-18 Procede d'orientation d'embryons vegetaux
CA002167501A CA2167501C (fr) 1992-04-08 1993-08-18 Methode pour l¨orientation directionnelle d¨embryons de plantes
NZ255823A NZ255823A (en) 1992-04-08 1993-08-18 Method of orienting plant embryos such that a specified end is always oriented in a specific direction; separation of conifer somatic embryos

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US07/865,390 US5284765A (en) 1992-04-08 1992-04-08 Method of directionally orienting plant embryos
BR9307878A BR9307878A (pt) 1992-04-08 1993-08-18 Processo para orientar direcionalmente uma multiplicidade dem embrioes de plantas
PCT/US1993/007804 WO1995005064A1 (fr) 1992-04-08 1993-08-18 Procede d'orientation d'embryons vegetaux
NZ255823A NZ255823A (en) 1992-04-08 1993-08-18 Method of orienting plant embryos such that a specified end is always oriented in a specific direction; separation of conifer somatic embryos

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NZ (1) NZ255823A (fr)
WO (1) WO1995005064A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001013702A3 (fr) * 1999-08-23 2001-08-16 Weyerhaeuser Co Systeme de delivrance d'embryons pour semences artificielles
US7131234B2 (en) 2003-11-25 2006-11-07 Weyerhaeuser Co. Combination end seal and restraint
US7228658B2 (en) 2003-08-27 2007-06-12 Weyerhaeuser Company Method of attaching an end seal to manufactured seeds
US7356965B2 (en) 2003-12-11 2008-04-15 Weyerhaeuser Co. Multi-embryo manufactured seed
US7530197B2 (en) 2003-06-30 2009-05-12 Weyerhaeuser Co. Automated system and method for harvesting and multi-stage screening of plant embryos
US7547488B2 (en) 2004-12-15 2009-06-16 Weyerhaeuser Nr Company Oriented strand board panel having improved strand alignment and a method for making the same
US7555865B2 (en) 2003-11-25 2009-07-07 Weyerhaeuser Nr Company Method and system of manufacturing artificial seed coats
US7568309B2 (en) 2004-06-30 2009-08-04 Weyerhaeuser Nr Company Method and system for producing manufactured seeds
US7591287B2 (en) 2003-12-18 2009-09-22 Weyerhaeuser Nr Company System and method for filling a seedcoat with a liquid to a selected level
US7603807B2 (en) 2003-11-26 2009-10-20 Weyerhaeuser Nr Company Vacuum pick-up device with mechanically assisted release
US7654037B2 (en) 2005-06-30 2010-02-02 Weyerhaeuser Nr Company Method to improve plant somatic embryo germination from manufactured seed

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US4957866A (en) * 1989-03-09 1990-09-18 Weyerhaeuser Company Method for reproducing coniferous plants by somatic embryogenesis
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US5036007A (en) * 1989-03-09 1991-07-30 Weyerhaeuser Company Method for reproducing coniferous plants by somatic embryogenesis using abscisic acid and osmotic potential variation
WO1992007457A1 (fr) * 1990-10-26 1992-05-14 Weyerhaeuser Company Analogues de graines botaniques
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WO1991000781A1 (fr) * 1989-07-06 1991-01-24 Valtion Teknillinen Tutkimuskeskus Procede et appareil de separation de corps de petites dimensions d'un liquide
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D.J. GRAY ET AL.: "Dessicated Quiescent Somatic Embryos of Orchardgrass for Use as Synthetic Seeds", IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY, vol. 23, no. 1, January 1987 (1987-01-01), pages 29 - 33 *
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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU765726B2 (en) * 1999-08-23 2003-09-25 Weyerhaeuser Company An embryo delivery system for manufactured seeds
US6684564B1 (en) 1999-08-23 2004-02-03 Weyerhaeuser Company Embryo delivery system for manufactured seeds
AU765726C (en) * 1999-08-23 2004-05-20 Weyerhaeuser Company An embryo delivery system for manufactured seeds
WO2001013702A3 (fr) * 1999-08-23 2001-08-16 Weyerhaeuser Co Systeme de delivrance d'embryons pour semences artificielles
US7530197B2 (en) 2003-06-30 2009-05-12 Weyerhaeuser Co. Automated system and method for harvesting and multi-stage screening of plant embryos
US7685767B2 (en) 2003-06-30 2010-03-30 Weyerhaeuser Nr Company Automated system and method for harvesting and multi-stage screening of plant embryos
US7228658B2 (en) 2003-08-27 2007-06-12 Weyerhaeuser Company Method of attaching an end seal to manufactured seeds
US7555865B2 (en) 2003-11-25 2009-07-07 Weyerhaeuser Nr Company Method and system of manufacturing artificial seed coats
US7131234B2 (en) 2003-11-25 2006-11-07 Weyerhaeuser Co. Combination end seal and restraint
US7603807B2 (en) 2003-11-26 2009-10-20 Weyerhaeuser Nr Company Vacuum pick-up device with mechanically assisted release
US7356965B2 (en) 2003-12-11 2008-04-15 Weyerhaeuser Co. Multi-embryo manufactured seed
US7591287B2 (en) 2003-12-18 2009-09-22 Weyerhaeuser Nr Company System and method for filling a seedcoat with a liquid to a selected level
US7568309B2 (en) 2004-06-30 2009-08-04 Weyerhaeuser Nr Company Method and system for producing manufactured seeds
US7547488B2 (en) 2004-12-15 2009-06-16 Weyerhaeuser Nr Company Oriented strand board panel having improved strand alignment and a method for making the same
US7654037B2 (en) 2005-06-30 2010-02-02 Weyerhaeuser Nr Company Method to improve plant somatic embryo germination from manufactured seed

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CA2167501C (fr) 1999-12-07
CA2167501A1 (fr) 1995-02-23

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