Liquid-based method for producing plant embryos

Abstract

The present invention relates to methods for developing embryos and producing germination-competent embryos using a liquid embryo development media.

1 .- 35 . (canceled) 
     
     
         36 . A method for determining an optimal settled cell volume for developing plant embryos comprising (1) adding a volume of proliferative plant cells to a volume of liquid embryonic development medium to produce a solution of cells in a vessel, (2) agitating the solution for a first period of time, (3) measuring cell density at least once during and/or after the first period of time, (4) further agitating the solution for a second period of time, (5) recording the number of embryos that have developed in the solution after the second period of time, (6) repeating steps (1) to (5) by adding a different volume of proliferative plant cells to the same volume of liquid embryonic development medium in another vessel and shaking and further shaking the solution for same periods of time, and (7) comparing the recorded numbers of plant embryos that have developed in the solutions, wherein the cell density value for the volume of proliferative plant cells that produces the most number of embryos is an optimal cell density for developing embryos from that species of plant cells. 
     
     
         37 . The method of  claim 36 , wherein the first period of time is about 1-3 weeks. 
     
     
         38 . The method of  claim 36 , wherein the second period of time is about 2 weeks. 
     
     
         39 . A method for determining an optimal settled cell volume for developing plant embryos comprising (1) adding a volume of proliferative plant cells to a volume of liquid embryonic development medium to produce a solution of cells, (2) agitating the solution for a first period of time, (3) removing an aliquot of the solution after the first period of time to a vessel, (4) allowing the cells in the aliquot of the solution to settle as a layer of cells, (5) measuring and recording the height of the cell layer to generate a settled cell volume (SCV) value, (6) further agitating the solution for a second period of time, (7) recording the number of embryos that have developed in the solution after the second period of time, (8) repeating steps (1) to (7) by adding a different volume of proliferative plant cells to the same volume of liquid embryonic development medium and shaking and further shaking the solution for same periods of time, and (9) comparing the recorded numbers of plant embryos that have developed in the solutions, wherein the SCV value for the volume of proliferative plant cells that produces the most number of embryos is an optimal SCV for developing embryos from that species of plant cells. 
     
     
         40 . A one-vessel method for developing plant embryos, comprising (1) incubating a volume of proliferative plant cells in a volume of liquid initiation medium in a vessel for a first period of time to enhance cell proliferation, (2) exchanging the volume of liquid proliferation medium after the first period of time with a volume of liquid embryonic development medium, and (3) incubating the proliferative plant cells in the liquid embryonic development medium for a second period of time in the same vessel, wherein plant embryos are developed after the second period of time, and wherein the liquid embryonic development medium comprises (i) phytohormone, (ii) a source of reduced nitrogen, and (iii) sugar. 
     
     
         41 . The method of  claim 40 , wherein the liquid embryonic development medium does not comprise a non-permeating osmotic agent. 
     
     
         42 . The method of  claim 41 , wherein the liquid embryonic development medium does not comprise polyethylene glycol. 
     
     
         43 . The method of  claim 40 , wherein the volume of liquid initiation medium in the vessel is about 1-50 liters. 
     
     
         44 . The method of  claim 40 , wherein the vessel is a bioreactor. 
     
     
         45 . The method of  claim 44 , wherein the bioreactor is a vessel, which comprises (1) a 2-20 liter-capacity bottle that comprises (i) a first port to supply air to the bottle, (ii) a second port to allow air to escape from the bottle, (iii) a third port for dispensing fresh medium or new medium from a reservoir into the bottle. 
     
     
         46 . The method of  claim 45 , wherein the step of exchanging the liquid initiation medium for the embryonic development medium in the bioreactor is performed under vacuum, whereby the liquid initiation medium is removed from the bottle and liquid embryonic development medium is drawn into the bottle from the reservoir via the third port. 
     
     
         47 . The method of  claim 40 , further comprising conditioning the developed embryos in the vessel for a period of time, wherein the embryos become germination-competent after the conditioning period. 
     
     
         48 . The method of  claim 47 , wherein the conditioning step entails removing the liquid embryonic development medium in the vessel and converting the atmosphere inside the vessel into a high relative humidity environment for the period of time. 
     
     
         49 . The method of  claim 48 , wherein the developed embryos are stored in the high relative humidity environment for 1-5 weeks. 
     
     
         50 . The method of  claim 40 , wherein the proliferative plant cells are incubated in the liquid embryonic development medium in the vessel for no more than 5 weeks. 
     
     
         51 .- 55 . (canceled) 
     
     
         56 . The method of  claim 40 , wherein the step of exchanging the liquid initiation medium for the embryonic development medium is automated.