Somatic embryogenesis and embryo harvesting and method for preparing plant embryos for plant production
View Patent ↗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 an embryonic tissue of a coniferous tree, comprising casein hydrolysate in a concentration from 2000 to about 3000 mg/l and activated carbon in an amount from 500 to 5000 mg/liter.
2. The medium of claim 1 , wherein the concentration of casein hydrolysate is from 2000 mg/l to 2500 mg/l.
3. The medium of claim 1 , further comprising myo-inositol, sucrose, and glutamine.
4. The medium of claim 3 , wherein the medium further comprises 2 to 30 g/l maltose, 3 mg/l 2,4-D, 0.5 mg/l BAP, 10 mg/l ABA, and 2 mg/l glycine; and either 3 g/l phytagel or 1.5 to 2 g/l gelrite.
5. The medium of claim 4 , further comprising brassinolide in an amount from 0.01 μM to 0.20 μM.
6. The medium of claim 1 , wherein the medium is liquid medium or gel medium.
7. 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.
8. The method of claim 7 , wherein the medium on which the proliferative somatic embryo tissue is cultured is liquid medium or gel medium.
9. The method of claim 8 , wherein the medium on which the proliferative somatic embryo tissue is cultured is liquid medium.
10. The method of claim 7 , wherein the step of harvesting the embryos further comprises rinsing the embryos with liquid to remove any polyethylene glycol adhered to the embryo surface.
11. The method of claim 10 , wherein the liquid is water.
12. The method of claim 7 , 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°degree. C.
13. The method of claim 7 , 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.
14. The method of claim 8 , wherein the liquid medium further comprises 0.01-1.00 μM of brassinolide.
15. The method of claim 13 , 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.
16. The method of claim 7 , wherein the high relative humidity environment is a sealed container.
17. The method of claim 16 , wherein the sealed container is a Petri dish.
18. The method of claim 7 , wherein the amount of polyethylene glycol in the maturation medium is from about 7% to about 13%.
19. The method of claim 7 , wherein the maturation medium comprises abscisic acid, glutamine, and maltose.
20. The method of claim 19 , wherein the maturation medium comprises the components listed in Table 1 for 2M21 in the specified amounts (mg/liter).
21. The method of claim 7 , 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.