somatic embryogenesis and embryo harvesting and method and apparatus for preparing plant embryos for plant production

Abstract

Described herein are methods and media for facilitating somatic embryogenesis and for collecting, conditioning, and transferring the washed embryos onto a substrate and into an environment suitable for conditioning the embryos for a desired period of time so they become germination-competent for plant production. The described plant embryo cleaning apparatus and method are used for preparing multiple plant embryos for plant production. The apparatus and method can use a cleaning fluid source, a fluid-conditioning system, a fluid-delivery structure, a cleaning station, an outlet mechanism, a negative pressure source, and a controller.

1 . A medium for growing embryonic tissue, comprising a high concentration of casein that is from about 1100 to about 3000 mg/l. 
     
     
         2 . The medium of  claim 1 , wherein the casein is casein hydrolysate. 
     
     
         3 . The medium of  claim 1 , wherein the concentration of casein is from about 2000 mg/l to about 2500 mg/l. 
     
     
         4 . The medium of  claim 1 , further comprising myo-inositol, sucrose, and glutamine. 
     
     
         5 . The medium of  claim 4 , wherein the medium further comprises the components listed in Table 1 for Mi3 in the specified amounts (mg/liter). 
     
     
         6 . The medium of  claim 5 , further comprising brassinolide. 
     
     
         7 . The method of  claim 6 , wherein the amount of brassinolide is 0.05 to 0.5 μM. 
     
     
         8 . The medium of  claim 1 , wherein the medium is liquid medium or gel medium. 
     
     
         9 . The medium of  claim 1 , wherein the medium is liquid medium. 
     
     
         10 . A method of producing germination ready somatic embryos, comprising:
 (A) culturing proliferative somatic embryo tissue in the medium of  claim 1 ;   (B) transferring the cultured tissue onto polyethylene glycol-containing maturation medium to produce embryos;   (C) harvesting the embryos that are produced from step (B); and   (D) placing the embryos in a high relative humidity environment for a period of time;   wherein after the period of time, the embryos are germination ready somatic embryos.   
     
     
         11 . The method of  claim 10 , wherein the medium on which the proliferative somatic embryo tissue is cultured is liquid medium or gel medium. 
     
     
         12 . The method of  claim 11 , wherein the medium on which the proliferative somatic embryo tissue is cultured is liquid medium. 
     
     
         13 . The method of  claim 10 , wherein the step of harvesting the embryos further comprises rinsing the embryos with liquid to remove any polyethylene glycol adhered to the embryo surface. 
     
     
         14 . The method of  claim 13 , wherein the liquid is water. 
     
     
         15 . The method of  claim 10 , wherein the period of time in which the embryos are placed in a high relative humidity environment is for either (a) about 1-5 weeks, or (b) 1-12 weeks at +4° C. 
     
     
         16 . The method of  claim 10 , further comprising the step of placing the harvested embryos on second maturation/conditioning medium for a second period of time prior to placement in the high relative humidity environment. 
     
     
         17 . The method of  claim 10 , wherein the liquid medium further comprises 0.01-1.00 μM of brassinolide. 
     
     
         18 . The method of  claim 16 , wherein the second period of time in which the harvested embryos are placed on the second medium is from 1 day to about 8 weeks. 
     
     
         19 . The method of  claim 10 , wherein the high relative humidity environment is a sealed container. 
     
     
         20 . The method of  claim 19 , wherein the sealed container is a Petri dish. 
     
     
         21 . The method of  claim 10 , wherein the amount of polyethylene glycol in the maturation medium is from about 7% to about 13%. 
     
     
         22 . The method of  claim 10 , wherein the maturation medium comprises abscisic acid, glutamine, and maltose. 
     
     
         23 . The method of  claim 22 , wherein the maturation medium comprises the components listed in Table 1 for 2M21 in the specified amounts (mg/liter). 
     
     
         24 . The method of  claim 10 , wherein the somatic embryo tissue is selected from the group consisting of Loblolly pine somatic embryo tissue, Pitch pine somatic embryo tissue, and Radiata pine somatic embryo tissue.