Search International and National Patent Collections
Some content of this application is unavailable at the moment.
If this situation persists, please contact us atFeedback&Contact
1. (WO1991005854) METHOD FOR REPRODUCING CONIFERS BY SOMATIC EMBRYOGENESIS
Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

CLAIMS:
1. A m e thod o f reproducing coni ferous pla n ts by so m atic embryogenesis which comprises:
placing a suitable explant on an culture medium containing sufficient amounts of nutrients and plant growth hormones to induce and grow a culture containing proembryos; and
transferring the proembryos to a cotyledonary embryo developm ent medium having a sufficient initial amount of exogenous abscisic acid and an adsorbent material for a sufficient tim e and under suitable environmental conditions to enable development of cotyledonary embryos, said adsorbent gradually reducing the level of available abscisic acid over tim e, said combination of abscisic acid and adsorbent yielding greater quantities of cotyledonary embryos of i mproved quality than media lacking the adsorbent material.

2. The method of claim 1 which further includes transferring the proembryos from the induction culture to an intermediate maintenance and multiplication culture medium having a significantly reduced level of plant growth hormones prior to transferring the proembryos to the cotyledonary embryo development medium.

3. The method of claims 1 or 2 which further include transferring the proembryos from the initiation or maintenance and m ultiplication media to a culture medium having a raised osmotic potential of at least about 170 mM/kg in order to induce late stage proembryo development prior to transferring the proe mbryos to the cotyledonary embryo development medium.

4. The method of claim 1 in which the adsorbent material in the developm ent mediu m is selected from the group consisting of activated charcoal, silica gel, activated alum ina, poly(vinylpyrrolidone), molecular sieves, and mixtures thereof.

5. The method of clai m 4 in which the adsorbent material is activated charcoal present in a range of about 0.1-5.0 g/L o f culture medium.

6. The method of claims 1 or 5 in which abscisic acid is initially present in a range of about 5-100 mg/L of culture medium.

7. The method of claim 1 in which the coniferous plant is Picea abies.

8. The method of claim 1 in which the coniferous plant is Pinus taeda.

9. The method of claim 1 in which the coniferous plant is

Pseudotsuga menziesii.

10. A method suitable for further developing tissue culture induced coniferous plant species somatic proembryos into well developed cotyledonary embryos which comprises:
transferring and further culturing the proembryos using a development medium having sufficient amounts of mineral and organic nutrient materials and a sufficient initial amount of abscisic acid along with an adsorbent material to gradually reduce the the level of available abscisic acid over time, said medium also having sufficient osmoticants to raise the osmotic potential to at least about 350 mM/kg, so as to enable and promote the development and growth of robust cotyledonary embryos having a high potential for normal germination and plant development.

11. The method of claim 10 which further includes transferring the proembryos from an induction culture medium to an intermediate maintenance and multiplication culture medium having a significantly reduced level of plant growth hormones prior to transferring the proembryos to the cotyledonary embryo development medium.

12. The method of claim 10 or 11 which further includes transferring the proembryos from the initiation or maintenance and multiplication media to a culture medium having a higher osmotic potential, said osmotic potential being least about 170 mM/kg, in order to induce late stage proembryo development prior to transferring the proembryos into the cotyledonary embryo development medium.

13. The method of claim 10 in which the adsorbent material in the development medium is selected from the group consisting of activated charcoal, silica gel, activated alu mina, poly(vinylpyrrolidone), molecular sieves, and mixtures thereof.

14. The method of claim 10 in which the osmotic potential of the development medium is controlled by a material combining a readily metabolized carbohydrate energy source and at least one additional osmoticant poorly metabolized by the developing embryos.

15. The m ethod of claim 14 in which the readily metabolized osmoticant is selected from the group consisting of sucrose, glucose, fructose, maltose, galactose, and mixtures thereof.

16. The m ethod of claim 14 in which the poorly metabolized osmoticant is selected from the group consisting of sorbitol, lactose, a polyalkylene glycol, and mixtures thereof.

17, The method of claim 10 in which the embryo culture growing in the development medium is transferred at least once to a fresh development medium containing osmoticants which may be different from those in the preceeding medium, said fresh development medium also having an osmotic potential which may differ from that of the preceeding medium.

18. The method of claim 10 in which the osmotic potential of the development medium is maintained above a level of about 400 mM/kg.

19. The method of claim 17 in which the osmotic potential of the development media are always maintained above a level of about 400 mM/kg.

20. The method of claim 17 in which the osmotic potential of each successive fresh developm ent m ediu m is raised over that of the preceeding development medium.

21. The method of claim 10 in which the plant is Douglas-fir (Pseudotsuga m enziesii) and the explant com prises the zygotic em bryo extracted from an im mature seed.

22. The method of claim 21 which includes, prior to transfer of proembryos to the development medium, the further step of transferring the proembryos to a liquid culture medium having a reduced osmotic potential and containing a sufficient amount of exogenous abscisic acid in order to cause singulation of said proembryos.

23. The method of claim 22 in which the osmotic potential of the singulation medium is below about 150 m M/kg.

24. The method of claim 22 in which the abscisic acid level in the singulation medium is initially in the range of about 5-15 ppm.

25. The m ethod of claim 22 in which at least one transfer to fresh medium is made during the singulation step, said fresh medium having a lower concentration of abscisic acid than that initially present in the previous m ediu m, said lower concentration being no greater than about that present in the previous medium im mediately prior to the transfer.

26. The method of claims 22, 23, 24, or 25 in which the initial available exogenous abscisic acid in the .developm ent m edium is no greater than that present in the singulation medium im mediately prior to transfer into the development medium singulation step.

27. The method of claim 26 in which the development medium lacks exogenous abscisic acid en t irely a nd a su f fic ien t a m ount o f abscisic acid is transferred with the embryos as entrained or endogenous abscisic acid from the singulation step.

28. The m ethod of clai m 22 in which the concentration of abscisic acid to which the embryos are exposed is reduced continuously from the beginning of the singulation period until the end of the development period.