Low density spreading methods for conifer somatic embryogenesis
View Patent ↗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.
1. A method of producing conifer cotyledonary somatic embryos, the method comprising:
(a) uniformly dispersing pre-cotyledonary somatic embryos onto a porous material horizontally disposed over a non-porous surface at a density of less than 0.05 gram wet cell weight of pre-cotyledonary somatic embryos per square inch of porous material by suspending the pre-cotyledonary embryos in a volume of sterile dilution medium sufficient to submerge the surface of the porous material and dispensing the pre-cotyledonary somatic embryos in the sterile dilution medium onto the porous material;
(b) removing the sterile dilution medium from the porous material, thereby trapping the uniformly dispersed pre-cotyledonary somatic embryos on the porous material; and
(c) contacting the pre-cotyledonary somatic embryos trapped on the porous material with development medium for a period of time sufficient to produce conifer cotyledonary somatic embryos.
2. The method of claim 1 , wherein the pre-cotyledonary somatic embryos are uniformly dispersed according to step (a) at a density of from 0.001 gram to 0.05 gram wet cell weight of pre-cotyledonary somatic embryos per square inch of porous material.
3. The method of claim 1 , wherein the porous material comprises pores having an average pore diameter in the range of from 5 microns to 1200 microns.
4. The method of claim 1 , wherein the porous material is non-absorbent.
5. The method of claim 3 , wherein the porous material is a woven mesh.
6. The method of claim 1 , wherein the porous material is attached to a support frame comprising handles.
7. The method of claim 6 , wherein the sterile dilution medium is removed from the porous material according to step (b) by vertically lifting the support frame off the non-porous surface.
8. The method of claim 1 , wherein the sterile dilution medium is removed from the porous material according to step (b) by reducing the sterile dilution medium to a level below the surface of the porous material.
9. The method of claim 1 , wherein the development medium according to step (c) is a liquid medium.
10. The method of claim 1 , wherein the development medium according to step (c) is a solid medium.
11. A method of producing conifer cotyledonary somatic embryos comprising:
(a) culturing conifer embryogenic cells in a liquid maintenance medium to form pre-cotyledonary conifer somatic embryos;
(b) uniformly dispersing the pre-cotyledonary somatic embryos prepared in step (a) onto a porous material horizontally disposed over a non-porous surface at a density of less than 0.05 gram wet cell weight of pre-cotyledonary somatic embryos per square inch of porous material by suspending the pre-cotyledonary embryos in a volume of sterile dilution medium sufficient to submerge the surface of the porous material and dispensing the pre-cotyledonary somatic embryos in the sterile dilution medium onto the porous material;
(c) removing the sterile dilution medium from the porous material, thereby trapping the uniformly dispersed pre-cotyledonary somatic embryos on the porous material; and
(d) contacting the pre-cotyledonary somatic embryos trapped on the porous material with development medium for a period of time sufficient to produce conifer cotyledonary somatic embryos.