NUTRITIVE MEDIA AND MANUFACTURED SEEDS COMPRISING SAME

MILIEUX NUTRITIFS ET GRAINES MANUFACTUREES COMPRENANT DE TELS MILIEUX NUTRITIFS

English Abstract

Manufactured seeds are disclosed that comprise a unit of totipotent plant
tissue and a nutritive medium. The nutritive medium can contain a number of
different components selected from the following: a gel solute, charcoal, a
carbon source, urea, KNO3, NH4NO3, CuC12, CuSO4, KI, KH2PO4, CaC12, MgSO4,
Na2EDTA, FeSO4, ferric citrate, MnSO4, MnCl2, H3BO3, ZnSO4, CoCl2, Na2MoO4,
(NH4)2MoO4, thiamine, riboflavin, pyridoxine. HCl, Ca-pantothenate, nicotinic
acid, biotin, folic acid, and myo-inositol. The nutritive medium can also
include any of various protein amino acids, any of various polyamines, any of
various oxygen-absorbing compounds, any of various non-protein amino acids,
and/or a smoke suspension.

French Abstract

Cette invention se rapporte à des graines manufacturées qui comprennent une unité faite d'un tissu végétal totipotent et d'un milieu nutritif. Ce milieu nutritif peut renfermer un certain nombre de différents constituants choisis parmi les constituants suivants: un soluté en gel, du charbon, une source de carbone, de l'urée, KNO¿3?, NH¿4?NO¿3?, CuCl¿2?, CuSo¿4?, KI, KH¿2?PO¿4?, CaCl¿2?, MgSO¿4?, Na¿2?EDTA, FeSO¿4?, du citrate ferrique, MnSO¿4?, MnCl¿2?, H¿3?BO¿3?, ZnSO¿4?, CoCl¿2?, Na¿2?MoO¿4?, (NH¿4?)¿2?MoO¿4?, thiamine, riboflavine, pyridoxine.HCl, Ca-pantothénate, de l'acide nicotinique, de la biotine, de l'acide folique et du myo-inositol. Pour constituer ce milieu nutritif, on peut également y ajouter quelques acides aminés protéinés choisis parmi une variété de ces acides aminés, quelques polyamines choisies parmi une variété de polyamines, quelques composés absorbant l'oxygène choisis parmi une variété de ces composés, quelques acides aminés non protéinés choisis parmi une variété de ces acides aminés, ainsi qu'une suspension de fumée.

Claims

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1. A manufactured seed comprising:
(a) a structure for containing a plant embryo; and
(b) a nutritive medium comprising about 5 mM to about 30 mM urea,
about 0.01 mM to about 8 mM L-arginine, and about 0.001 mM to
about 0.01 mM thiamine-HCl wherein the nutritive medium is
positioned to be capable of functional contact with at least a root end
of the plant embryo.
2. The manufactured seed according to claim 1, wherein the urea
concentration is about 5 mM to about 20 mM.
3. The manufactured seed according to claim 1 or 2, wherein the
urea concentration is about 13.32 mM.
4. The manufactured seed according to any one of claims 1 through
3, wherein the arginine concentration is about 0.01 mM to about 4 mM.
5. The manufactured seed according to any one of claims 1 through
4, wherein the arginine concentration is about 1.0471 mM.
6. The manufactured seed according to any one of claims 1 through
5, wherein the thiamine-HCl concentration is about 0.001 mM to about 0.005
mM.
7. The manufactured seed according to any one of claims 1 through
6, wherein the thiamine-HCl concentration is about 0.003 mM.
8. The manufactured seed according to any one of claims 1 through
7, wherein the nutritive medium further comprises charcoal.

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9. The manufactured seed according to claim 8, wherein the
charcoal concentration is about 2 g/L to 5 g/L.
10. The manufactured seed according to claim 9, wherein the
charcoal concentration is about 2 g/L to 3 g/L.
11. The manufactured seed according to claim 10, wherein the
charcoal concentration is about 2.5 g/L.
12. The manufactured seed according to any one of claims 1 through
11, wherein the nutritive medium further comprises a non-protein amino acid.
13. The manufactured seed according to claim 12, wherein the non-
protein amino acid concentration is about 0.1 mM to 3 mM.
14. The manufactured seed according to claim 13, wherein the non-
protein amino acid concentration is about 0.1 mM to 1.25 mM.
15. The manufactured seed according to any one of claims 12
through 14, wherein the non-protein amino acid is ornithine.
16. The manufactured seed according to any one of claims 12
through 14, wherein the non-protein amino acid is selected from the group
consisting of arginosuccinate, citrulline, and canavanine.
17. The manufactured seed according to any one of claims 1 through
16, wherein the nutritive medium further comprises sucrose.
18. The manufactured seed according to claim 17, wherein the
sucrose concentration is about 146.07 mM.

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19. The manufactured seed according to claim 17, wherein the
sucrose concentration is about 58.43 g/L.
20. The manufactured seed according to any one of claims 1 through
19, wherein the nutritive medium further comprises a smoke suspension.
21. The manufactured seed according to claim 20, wherein the smoke
suspension concentration is about 0.1 mL/L medium to 10 mL/L medium.
22. The manufactured seed according to claim 21, wherein the smoke
suspension concentration is about 0.1 mL/L medium to 1 mL/L medium.
23. The manufactured seed according to any one of claims 1 through
22, wherein the nutritive medium further comprises a polyamine selected from
the group consisting of putrescine, spermidine, spermine, and mixtures
thereof.
24. The manufactured seed according to claim 23, wherein the
polyamine concentration is about 0.1 mM to 0.5 mM.
25. The manufactured seed according to claim 1, wherein the
nutritive medium further comprises at least one component selected from the
group consisting of about 0.01 mM to 2 mM L-asparagine, 0.01 mM to 6 mM L-
glutamine, 0.01 mM to 2 mM L-lysine, 0.01 mM to 2 mM L-serine, 0.01 mM to
2 mM L-proline, 0.01 mM to 2 mM L-valine, 0.01 mM to 2 mM L-alanine, 0.01
mM to 2 mM L-cysteine, 0.01 mM to 2 mM L-leucine, 0.01 mM to 2 mM L-
tyrosine, 0.01 mM to 2 mM L-threonine, 0.01 mM to 2 mM L-phenylalanine,
0.01 mM to 2 mM L-histidine, 0.002 mM to 0.2 mM L-glycine, 0.01 mM to 2
mM L-tryptophan, 0.01 mM to 2 mM L-isoleucine and 0.01 mM to 2 mM L-
methionine.

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26. The manufactured seed according to claim 1, wherein the
nutritive medium further comprises at least one component selected from the
group consisting of about 0.01 mM to 1 mM L-asparagine, 0.01 mM to 3 mM L-
g1 utamine, 0.01 mM to 1 mM L-lysine, 0.01 mM to 1 mM L-serine, 0.01 mM to
1 mM L-proline, 0.01 mM to 1 mM L-valine, 0.01 mM to 1 mM L-alanine, 0.01
mM to 1 mM L-cysteine, 0.01 mM to 1 mM L-leucine, 0.01 mM to 1 mM L-
tyrosine, 0.01 mM to I mM L-threonine, 0.01 mM to 1 mM L-phenylalanine,
0.01 mM to 1 mM L-histidine, 0.01 mM to 0.1 mM L-glycine, 0.01 mM to 1 mM
L-tryptophan, 0.01 mM to 1 mM L-isoleucine and 0.01 mM to 1 mM L-
methionine.
27. The manufactured seed according to claim 1wherein the nutritive
medium further comprises at least one component selected from the group
consisting of about 0.8076 mM L-asparagine, 1.8248 mM L-glutamine, 0.1624
mM L-lysine, 0.7613 mM L-serine, 0.463 mM L-proline, 0.455 mM L-valine,
0.5983 mM L-alanine, 0.2204 mM L-cysteine, 0.6099 mM L-leucine, 0.2942
mM L-tyrosine, 0.2241 mM L-threonine, 0.3227 mM L-phenylalanine, 0.1721
mM L-histidine, 0.71 mM L-glycine, 0.1307 mM L-tryptophan, 0.2036 mM L-
isoleucine, and 0.1789 mM L-methionine.
28. The manufactured seed according to claim 1, wherein the
nutritive medium further comprises at least one component selected from the
group consisting of about 10 g/L to 30 g/L gel solute, 5 mM to 200 mM carbon
source, 10 mM to 20 mM KNO3, 2.5 mM to 5 mM NH4NO3, 0.002 mM to 0.01
mM KI, 0.01 mM to 20 mM KH2PO4, 0.5 mM to 5 mM CaCl2, 0.1 mM to 10 mM
MgSO4, 0.01 mM to 0.1 mM Na2EDTA, 0.01 mM to 0.1 mM FeSO4, 0.1 mM to
0.5 mM ferric citrate, 0.02 mM to 0.1 mM MnSO4, 0.01 mM to 0.1 mM MnCl2,
0.01 mM to 1 mM H3BO3, 0.0001 mM to 0.005 mM ZnSO4, 1x10 -5 mM to 1x10-
4 mM CoCl2, 0.001 mM to 0.005 mM CuCl2, 1x10 -5 mM to 1x10 -4 mM CuSO4,
1x10 -4 mM to 0.001 mM Na2MoO4, 1x10 -4 mM to 0.001 mM (NH4)2MoO4, 1x10-
4 mM to 0.001 mM Riboflavin, 0.001 mM to 0.005mM pyridoxine.cndot.HCl, 0.0005

25
mM to 0.004 mM Ca-pantothenate, 0.001 mM to 0.02 mM nicotinic acid, 1x10 -7
mM to 1x10 -5 mM Biotin, 1x10 -4 mM to 1x10 -3 mM folic acid, and 0.1 mM to
2.5
mM myo-inositol.
29. The manufactured seed according to claim 1, wherein the
nutritive medium further comprises at least one component selected from the
group consisting of about 10 g/L to 25 g/L gel solute, 25 mM to 175 mM
carbon source, 10 mM to 15 mM KNO3, 2 mM to 4 mM NH4NO3, 0.002 mM to
0.005 mM KI, 0.05 mM to 18 mM KH2PO4, 1 mM to 3 mM CaCl2, 0.2 mM to 3
mM MgSO4, 0.01 mM to 0.8 mM Na2EDTA, 0.01 mM to 0.05 mM FeSO4, 0.1
mM to 0.4 mM ferric citrate, 0.02 mM to 0.5 mM MnSO4, 0.01 mM to 0.05 mM
MnCl2, 0.1 mM to 0.5 mM H3BO3, 0.0003 mM to 0.004 mM ZnSO4, 0.001 mM
to 0.004 mM pyridoxine-HCl, 0.0005 mM to 0.015 mM Ca-pantothenate, 0.001
mM to 0.01 mM nicotinic acid, and 0.1 mM to 1 mM myo-inositol.
30. The manufactured seed according to claim 1, wherein the
nutritive medium further comprises at least one component selected from the
group consisting of about 3.74 mM NH4NO3, 13.23 mM KH2PO4, 1.54 mM
CaCl2, 1.98 mM MgSO4, 0.245 mM ferric citrate, 0.019 mM MnCl2, 0.162 mM
H3B03, 0.0023 mM CuCl2, 0.0003 mM (NH4)2MoO4, 0.1 mg/L GA4/7, 0.0003
mM Riboflavin, 0.0012 mM pyridoxine.cndot.HCl, 0.001 mM Ca-pantothenate,
0.0081 mM nicotinic acid, 4.09x10 -6 Biotin, 0.0003 mM folic acid, and 0.555
mM myo-inositol.
31. The manufactured seed according to claim 1, wherein the
nutritive medium further comprises at least one component selected from the
group consisting of about 11.57 mM KNO3, 2.577 mM NH4NO3, 0.0025 mM KI,
0.625 mM KH2PO4, 1.133 mM CaCl2, 0.367 mM MgSO4, 0.05 mM Na2EDTA,
0.027 mM FeSO4, 0.045 mM MnSO4, 0.05 mM H3BO3, 8.4x10 -3 mM ZnSO4,
3.2x10 -5 mM CuSO4, 2.9x10 -5 mM CoCl2, 4.4x10 -4 mM Na2MoO4, 0.0024 mM

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pyridoxine.cndot.HCl, 0.0041 mM nicotinic acid, 0.555 mM myo-inositol, and
0.027
mM L-glycine.
32. The manufactured seed according to any one of claims 1 through
31, wherein the nutritive medium further comprises at least one component
selected from the group consisting of about 18 g/L agar, 3.6 g/L Pluronic F-
68,
and 240 mL/L DC-200 silicone oil.
33. The manufactured seed according to any one of claims 1 through
32, wherein the pH of nutritive medium is about 5.7.
34. The manufactured seed according to any one of claims 1 through
33 wherein the plant embryo is a gymnosperm plant embryo.
35. The manufactured seed according to claim 34 wherein the
gymnosperm is Pine.
36. A method for germinating a plant embryo, comprising placing at
least a root end the plant embryo in functional contact with a nutritive
medium
comprising about 5 mM to about 30 mM urea, about 0.01 mM to about 8 mM
L-arginine, and about 0.001 mM to about 0.01 mM thiamine-HCl in a
manufactured seed.
37. The method according to claim 36, wherein the urea
concentration is about 5 mM to about 20 mM.
38. The method according to claim 36 or 37, wherein the urea
concentration is about 13.32 mM.
39. The method according to any one of claims 36 through 38,
wherein the arginine concentration is about 0.01 mM to about 4 mM.

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40. The method according to any one of claims 36 through 39,
wherein the arginine concentration is about 1.0471 mM.
41. The method according to any one of claims 36 through 40,
wherein the thiamine-HCl concentration is about 0.001 mM to about 0.005
mM.
42. The method according to any one of claims 36 through 41,
wherein the thiamine-HCl concentration is about 0.003 mM.
43. The method according to any one of claims 36 through 42,
wherein the nutritive medium further comprises charcoal.
44. The method according to claim 43, wherein the charcoal
concentration is about 2 g/L to 5 g/L.
45. The method according to claim 44, wherein the charcoal
concentration is about 2 g/L to 3 g/L.
46. The method according to claim 45, wherein the charcoal
concentration is about 2.5 g/L.
47. The method according to any one of claims 36 through 46,
wherein the nutritive medium further comprises a non-protein amino acid.
48. The method according to claim 47, wherein the non-protein amino
acid concentration is about 0.1 mM to 3 mM.
49. The method according to claim 48, wherein the non-protein amino
acid concentration is about 0.1 mM to 1.25 mM.

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50. The method according to any one of claims 47 through 49,
wherein the non-protein amino acid is ornithine.
51. The method according to any one of claims 47 through 49,
wherein the non-protein amino acid is selected from the group consisting of
arginosuccinate, citrulline, and canavanine.
52. The method according to any one of claims 36 through 51,
wherein the nutritive medium further comprises sucrose.
53. The method according to claim 52, wherein the sucrose
concentration is about 146.07 mM.
54. The method according to claim 52, wherein the sucrose
concentration is about 58.43 g/L.
55. The method according to any one of claims 36 through 54,
wherein the nutritive medium further comprises a smoke suspension.
56. The method according to claim 55, wherein the smoke
suspension concentration is about 0.1 mL/L medium to 10 mL/L medium.
57. The method according to claim 56, wherein the smoke
suspension concentration is about 0.1 mL/L medium to 1 mL/L medium.
58. The method according to any one of claims 36 through 57,
wherein the nutritive medium further comprises a polyamine selected from the
group consisting of putrescine, spermidine, spermine, and mixtures thereof.
59. The method according to claim 58, wherein the polyamine
concentration is about 0.1 mM to 0.5 mM.

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60. The method according to claim 36, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 0.01 mM to 2 mM L-asparagine, 0.01 mM to 6 mM L-glutamine, 0.01
mM to 2 mM L-lysine, 0.01 mM to 2 mM L-serine, 0.01 mM to 2 mM L-proline,
0.01 mM to 2 mM L-valine, 0.01 mM to 2 mM L-alanine, 0.01 mM to 2 mM L-
cysteine, 0.01 mM to 2 mM L-leucine, 0.01 mM to 2 mM L-tyrosine, 0.01 mM
to 2 mM L-threonine, 0.01 mM to 2 mM L-phenylalanine, 0.01 mM to 2 mM L-
histidine, 0.002 mM to 0.2 mM L-glycine, 0.01 mM to 2 mM L-tryptophan, 0.01
mM to 2 mM L-isoleucine and 0.01 mM to 2 mM L-methionine
61. The method according to claim 36, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 0.01 mM to 1 mM L-asparagine, 0.01 mM to 3 mM L-glutamine, 0.01
mM to 1 mM L-lysine, 0.01 mM to 1 mM L-serine, 0.01 mM to 1 mM L-proline,
0.01 mM to 1 mM L-valine, 0.01 mM to 1 mM L-alanine, 0.01 mM to 1 mM L-
cysteine, 0.01 mM to 1 mM L-leucine, 0.01 mM to 1 mM L-tyrosine, 0.01 mM
to I mM L-threonine, 0.01 mM to 1 mM L-phenylalanine, 0.01 mM to 1 mM L-
histidine, 0.01 mM to 0.1 mM L-glycine, 0.01 mM to 1 mM L-tryptophan, 0.01
mM to 1 mM L-isoleucine and 0.01 mM to 1 mM L-methionine.
62. The method according to claim 36, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 0.8076 mM L-asparagine, 1.8248 mM L-glutamine, 0.1624 mM L-
lysine, 0.7613 mM L-serine, 0.463 mM L-proline, 0.455 mM L-valine, 0.5983
mM L-alanine, 0.2204 mM L-cysteine, 0.6099 mM L-leucine, 0.2942 mM L-
tyrosine, 0.2241 mM L-threonine, 0.3227 mM L-phenylalanine, 0.1721 mM L-
histidine, 0.71 mM L-glycine, 0.1307 mM L-tryptophan, 0.2036 mM L-
isoleucine, and 0.1789 mM L-methionine.
63. The method according to claim 36, wherein the nutritive medium
further comprises at least one component selected from the group consisting

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of about 10 g/L to 30 g/L gel solute, 5 mM to 200 mM carbon source, 10 mM
to 20 mM KNO3, 2.5 mM to 5 mM NH4NO3, 0.002 mM to 0.01 mM KI, 0.01 mM
to 20 mM KH2PO4, 0.5 mM to 5 mM CaCl2, 0.1 mM to 10 mM MgSO4, 0.01 mM
to 0.1 mM Na2EDTA, 0.01 mM to 0.1 mM FeSO4, 0.1 mM to 0.5 mM ferric
citrate, 0.02 mM to 0.1 mM MnSO4, 0.01 mM to 0.1 mM MnCl2, 0.01 mM to 1
mM H3BO3, 0.0001 mM to 0.005 mM ZnSO4, 1x10 -5 mM to 1x10 -4 mM CoCl2,
0.001 mM to 0.005 mM CuCl2, 1x10 -5 mM to 1x10 -4 mM CuSO4, 1x10 -4 mM to
0.001 mM Na2MoO4, 1x10 -4 mM to 0.001 mM (NH4)2MoO4, 1x10 -4 mM to 0.001
mM Riboflavin, 0.001 mM to 0.005mM pyridoxine.cndot.HCl, 0.0005 mM to 0.004
mM Ca-pantothenate, 0.001 mM to 0.02 mM nicotinic acid, 1x10 -7 mM to 1x10 -
mM Biotin, 1x10 -4 mM to 1x10 -3 mM folic acid, and 0.1 mM to 2.5 mM myo-
inositol.
64. The method according to claim 36, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 10 g/L to 25 g/L gel solute, 25 mM to 175 mM carbon source, 10 mM
to 15 mM KNO3, 2 mM to 4 mM NH4NO3, 0.002 mM to 0.005 mM KI, 0.05 mM
to 18 mM KH2PO4, 1 mM to 3 mM CaCl2, 0.2 mM to 3 mM MgSO4, 0.01 mM to
0.8 mM Na2EDTA, 0.01 mM to 0.05 mM FeSO4, 0.1 mM to 0.4 mM ferric
citrate, 0.02 mM to 0.5 mM MnSO4, 0.01 mM to 0.05 mM MnCl2, 0.1 mM to 0.5
mM H3BO3, 0.0003 mM to 0.004 mM ZnSO4, 0.001 mM to 0.004 mM
pyridoxine.cndot.HCl, 0.0005 mM to 0.015 mM Ca-pantothenate, 0.001 mM to 0.01
mM nicotinic acid, and 0.1 mM to 1 mM myo-inositol.
65. The method according to claim 36, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 3.74 mM NH4NO3, 13.23 mM KH2PO4, 1.54 mM CaCl2, 1.98 mM
MgSO4, 0.245 mM ferric citrate, 0.019 mM MnCl2, 0.162 mM H3BO3, 0.0023
mM CuCl2, 0.0003 mM (NH4)2MoO4, 0.1 mg/L GA4/7, 0.0003 mM Riboflavin,
0.0012 mM pyridoxine.cndot.HCl, 0.001 mM Ca-pantothenate, 0.0081 mM nicotinic
acid, 4.09x10 -6 Biotin, 0.0003 mM folic acid, and 0.555 mM myo-inositol.

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66. The method according to claim 36, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 11.57 mM KNO3, 2.577 mM NH4NO3, 0.0025 mM KI, 0.625 mM
KH2PO4, 1.133 mM CaCl2, 0.367 mM MgSO4, 0.05 mM Na2EDTA, 0.027 mM
FeSO4, 0.045 mM MnSO4, 0.05 mM H3B03, 8.4x10 -3 mM ZnSO4, 3.2x10 -5 mM
CuSO4, 2.9x10 -5 mM CoCl2, 4.4x10 -4 mM Na2MoO4, 0.0024 mM
pyridoxine.cndot.HCl, 0.0041 mM nicotinic acid, 0.555 mM myo-inositol, and
0.027
mM L-glycine.
67. The method according to any one of claims 36 through 66,
wherein the nutritive medium further comprises at least one component
selected from the group consisting of about 18 g/L agar, 3.6 g/L Pluronic F-
68,
and 240 mL/L DC-200 silicone oil.
68. The method according to any one of claims 36 through 67,
wherein the pH of nutritive medium is about 5.7.
69. A method for germinating a plant embryo comprising placing the plant
embryo in a manufactured seed according to any one of claims 1 through 35.
70. The use of a nutritive medium for germinating a plant embryo by
placing the nutritive medium into functional contact with at least a root end
of the
plant embryo, the nutritive medium comprising about 5 mM to about 30 mM urea,
about 0.01 mM to about 8 mM L-arginine, and about 0.001 mM to about 0.01 mM
thiamine-HCl in a manufactured seed.
71. The use according to claim 70, wherein the urea concentration is
about 5 mM to about 20 mM.
72. The use according to claim 70 or 71, wherein the urea
concentration is about 13.32 mM.

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73. The use according to any one of claims 70 through 72, wherein
the arginine concentration is about 0.01 mM to about 4 mM.
74. The use according to any one of claims 70 through 73, wherein
the arginine concentration is about 1.0471 mM.
75. The use according to any one of claims 70 through 74, wherein
the thiamine-HCl concentration is about 0.001 mM to about 0.005 mM.
76. The use according to any one of claims 70 through 75, wherein
the thiamine-HCl concentration is about 0.003 mM.
77. The use according to any one of claims 70 through 76, wherein
the nutritive medium further comprises charcoal.
78. The use according to claim 77, wherein the charcoal
concentration is about 2 g/L to 5 g/L.
79. The use according to claim 78, wherein the charcoal
concentration is about 2 g/L to 3 g/L.
80. The use according to claim 79, wherein the charcoal
concentration is about 2.5 g/L.
81. The use according to any one of claims 70 through 80, wherein
the nutritive medium further comprises a non-protein amino acid.
82. The use according to claim 81, wherein the non-protein amino
acid concentration is about 0.1 mM to 3 mM.

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83. The use according to claim 82, wherein the non-protein amino
acid concentration is about 0.1 mM to 1.25 mM.
84. The use according to any one of claims 81 through 83, wherein
the non-protein amino acid is ornithine.
85. The use according to any one of claims 81 through 83, wherein
the non-protein amino acid is selected from the group consisting of
arginosuccinate, citrulline, and canavanine.
86. The use according to any one of claims 70 through 85, wherein
the nutritive medium further comprises sucrose.
87. The use according to claim 86, wherein the sucrose concentration
is about 146.07 mM.
88. The use according to claim 86, wherein the sucrose concentration
is about 58.43 g/L.
89. The use according to any one of claims 70 through 88, wherein
the nutritive medium further comprises a smoke suspension.
90. The use according to claim 189, wherein the smoke suspension
concentration is about 0.1 mL/L medium to 10 mL/L medium.
91. The use according to claim 90, wherein the smoke suspension
concentration is about 0.1 mL/L medium to 1 mL/L medium.
92. The use according to any one of claims 70 through 91, wherein
the nutritive medium further comprises a polyamine selected from the group
consisting of putrescine, spermidine, spermine, and mixtures thereof.

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93. The use according to claim 92, wherein the polyamine
concentration is about 0.1 mM to 0.5 mM.
94. The use according to claim 70, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 0.01 mM to 2 mM L-asparagine, 0.01 mM to 6 mM L-glutamine, 0.01
mM to 2 mM L-lysine, 0.01 mM to 2 mM L-serine, 0.01 mM to 2 mM L-proline,
0.01 mM to 2 mM L-valine, 0.01 mM to 2 mM L-alanine, 0.01 mM to 2 mM L-
cysteine, 0.01 mM to 2 mM L-leucine, 0.01 mM to 2 mM L-tyrosine, 0.01 mM
to 2 mM L-threonine, 0.01 mM to 2 mM L-phenylalanine, 0.01 mM to 2 mM L-
histidine, 0.002 mM to 0.2 mM L-glycine, 0.01 mM to 2 mM L-tryptophan, 0.01
mM to 2 mM L-isoleucine and 0.01 mM to 2 mM L-methionine.
95. The use according to claim 70, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 0.01 mM to 1 mM L-asparagine, 0.01 mM to 3 mM L-glutamine, 0.01
mM to 1 mM L-lysine, 0.01 mM to 1 mM L-serine, 0.01 mM to 1 mM L-proline,
0.01 mM to 1 mM L-valine, 0.01 mM to 1 mM L-alanine, 0.01 mM to 1 mM L-
cysteine, 0.01 mM to 1 mM L-leucine, 0.01 mM to 1 mM L-tyrosine, 0.01 mM
to I mM L-threonine, 0.01 mM to 1 mM L-phenylalanine, 0.01 mM to 1 mM L-
histidine, 0.01 mM to 0.1 mM L-glycine, 0.01 mM to 1 mM L-tryptophan, 0.01
mM to 1 mM L-isoleucine and 0.01 mM to 1 mM L-methionine.
96. The use according to claim 70, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 0.8076 mM L-asparagine, 1.8248 mM L-glutamine, 0.1624 mM L-
lysine, 0.7613 mM L-serine, 0.463 mM L-proline, 0.455 mM L-valine, 0.5983
mM L-alanine, 0.2204 mM L-cysteine, 0.6099 mM L-leucine, 0.2942 mM L-
tyrosine, 0.2241 mM L-threonine, 0.3227 mM L-phenylalanine, 0.1721 mM L-
histidine, 0.71 mM L-glycine, 0.1307 mM L-tryptophan, 0.2036 mM L-
isoleucine, and 0.1789 mM L-methionine.

35
97. The use according to claim 70, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 10 g/L to 30 g/L gel solute, 5 mM to 200 mM carbon source, 10 mM
to 20 mM KNO3, 2.5 mM to 5 mM NH4NO3, 0.002 mM to 0.01 mM KI, 0.01 mM
to 20 mM KH2PO4, 0.5 mM to 5 mM CaCl2, 0.1 mM to 10 mM MgSO4, 0.01 mM
to 0.1 mM Na2EDTA, 0.01 mM to 0.1 mM FeSO4, 0.1 mM to 0.5 mM ferric
citrate, 0.02 mM to 0.1 mM MnSO4, 0.01 mM to 0.1 mM MnCl2, 0.01 mM to 1
mM H3B03, 0.0001 mM to 0.005 mM ZnSO4, 1x10 -5 mM to 1x10 -4 mM CoCl2,
0.001 mM to 0.005 mM CuCl2, 1x10 -5 mM to 1x10 -4 mM CuSO4, 1x10 -4 mM to
0.001 mM Na2MoO4, 1x10 -4 mM to 0.001 mM (NH4)2MoO4, 1x10 -4 mM to 0.001
mM Riboflavin, 0.001 mM to 0.005mM pyridoxine.cndot.HCl, 0.0005 mM to 0.004
mM Ca-pantothenate, 0.001 mM to 0.02 mM nicotinic acid, 1x10 -7 mM to 1x10 -
mM Biotin, 1x10 -4 mM to 1x10 -3 mM folic acid, and 0.1 mM to 2.5 mM myo-
inositol.
98. The use according to claim 70, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 10 g/L to 25 g/L gel solute, 25 mM to 175 mM carbon source, 10 mM
to 15 mM KNO3, 2 mM to 4 mM NH4NO3, 0.002 mM to 0.005 mM KI, 0.05 mM
to 18 mM KH2PO4, 1 mM to 3 mM CaCl2, 0.2 mM to 3 mM MgSO4, 0.01 mM to
0.8 mM Na2EDTA, 0.01 mM to 0.05 mM FeSO4, 0.1 mM to 0.4 mM ferric
citrate, 0.02 mM to 0.5 mM MnSO4, 0.01 mM to 0.05 mM MnCl2, 0.1 mM to 0.5
mM H3BO3, 0.0003 mM to 0.004 mM ZnSO4, 0.001 mM to 0.004 mM
pyridoxine.cndot.HCl, 0.0005 mM to 0.015 mM Ca-pantothenate, 0.001 mM to 0.01
mM nicotinic acid, and 0.1 mM to 1 mM myo-inositol.
99. The use according to claim 70, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 3.74 mM NH4NO3, 13.23 mM KH2PO4, 1.54 mM CaCl2, 1.98 mM
MgSO4, 0.245 mM ferric citrate, 0.019 mM MnCl2, 0.162 mM H3BO3, 0.0023
mM CuCl2, 0.0003 mM (NH4)2MoO4, 0.1 mg/L GA4/7, 0.0003 mM Riboflavin,

36
0.0012 mM pyridoxine.cndot.HCl, 0.001 mM Ca-pantothenate, 0.0081 mM nicotinic
acid, 4.09x10 -6 Biotin, 0.0003 mM folic acid, and 0.555 mM myo-inositol.
100. The use according to claim 70, wherein the nutritive medium
further comprises at least one component selected from the group consisting
of about 11.57 mM KNO3, 2.577 mM NH4NO3, 0.0025 mM KI, 0.625 mM
KH2PO4, 1.133 mM CaCl2, 0.367 mM MgSO4, 0.05 mM Na2EDTA, 0.027 mM
FeSO4, 0.045 mM MnSO4, 0.05 mM H3BO3, 8.4x10 -3 mM ZnSO4, 3.2x10 -5 mM
CuSO4, 2.9x10 -5 mM CoCl2, 4.4x10 -4 mM Na2MoO4, 0.0024 mM
pyridoxine.cndot.HCl, 0.0041 mM nicotinic acid, 0.555 mM myo-inositol, and
0.027
mM L-glycine.
101. The use according to any one of claims 70 through 100, wherein
the nutritive medium further comprises at least one component selected from
the group consisting of about 18 g/L agar, 3.6 g/L Pluronic F-68, and 240 mL/L
DC-200 silicone oil.
102. The use according to any one of claims 70 through 101, wherein
the pH of nutritive medium is about 5.7.
103. The use according to any one of claims 70 through 102 wherein
the plant embryo is a gymnosperm plant embryo.
104. The use according to claim 103 wherein the gymnosperm is Pine.

Description

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NUTRITIVE MEDIA AND MANUFACTURED SEEDS COMPRISING SAME
FIELD OF THE INVENTION
The invention pertains to nutritive media, methods for using such media in the
production of manufactured seeds, and manufactured seeds comprising same.
BACKGROUND OF THE INVENTION
In many instances it is desirable to grow large numbers of genetically
identical plants.
These plants can be selected and grown based on their particular qualities,
such as their ability to
grow in a particular climate, or their ability to produce a particular type or
quality of fiber.
Unfortunately, in many cases the production of such plants through standard
breeding is not
feasible.
Standard plant breeding techniques are labor intensive and, because they
usually
involve fusion of gametes, tend to introduce genetic variability. The genetic
variability is a result
of crossing over of chromosomes, which often occurs during meiosis.
Additionally, standard
breeding techniques require that the breeder wait until a plant is mature
enough to breed. In some
cases, this means waiting many years. This delay decreases productivity and
increases costs.
Therefore, it is desirable to produce large numbers of genetically identical
plants via culturing of
somatic or zygotic plant embryos.
Somatic or zygotic plant embryos may be cultured in the form of "manufactured
seeds". Manufactured seeds are essentially analogs of botanic seed and
typically include a.nutrient
medium (termed here in a "nutritive medium"), and typically include structural
features that serve
to protect the embryo before, during, and after germination. References that
describe
manufactured seeds include U.S. Patent Nos. 5,564,224; 5,687,504; and
5,701,699, all to Carlson
et al. These patents disclose elements of such seeds, for instance, a
manufactured seed coat,
methods for using such manufactured seeds, and plant germinants produced from
manufactured
seeds.
A problem with manufactured seeds to date is the relatively low numbers of
successful germinants from such seeds compared to botanical (natural) seeds,
and relatively low
numbers of "normal" germinants from manufactured seeds, i.e., viable, uniform,
and
commercially useful germinants. Abnormal germinants possess any of various
malformations such
as small and/or deformed radicals, hypocotyls, or cotyledons. Although many
factors appear to
cause abnormal germination, the results generally indicate that manufactured
seeds, as currently
known in the art, do not provide a correct balance of nutrients and other
elements to the seed.
Therefore, there is a need for nutritive media that contain a suitable profile
of
nutrients and other elements that, when incorporated into manufactured seeds,
facilitate normal and
successful germination and germinant growth with normal organ development.

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In order to be commercially viable, a nutritive medium desirably facilitates
the
germination of manufactured seeds at a rate at least similar to the
germination rate of sexually
produced seeds. Commercial viability can be assessed by taking into account
the market size and
the volume of product necessary to meet market needs. In the case of plant-
related products the
market is vast. Therefore, a difference in the germination rate of 10% or 20%
can have an
immense impact on the commercial viability of manufactured seeds.
Researchers have investigated the effects caused by the addition of various
compounds to nutritive media in order to further the understanding of
germination and/or growth of
plant embryos on such media. For example, the effect of various nutrients on
chlorophyll
formation in embryos grown in the dark has been studied by Bogorad, Botanical
Gazette 3: 221-
241, 1950, and by Engvild, Physiologia Plantarum 17: 866-874, 1964; the effect
of sucrose
concentration has been studied by Schau, Physiologia Plantarum 4: 617-620,
1951, and by Ball,
American Journal of Botany 46: 130-139, 1958; the effect of cytokinins on
germination has been
studied by Khan, Science 71:853-859, 1971; the effect of smoke on germination
has been studied
by Brown and Van Staden, Plant Growth Regulation 22:115-124, 1997; and the
effect of amino
acids as sources of organic nitrogen has been studied by Engvild (cited
above).
In the laboratory, growth of plant embryos on the surface of an agar medium in
a
petri dish or the like is known. Unfortunately, the correlation between the
germination of a plant
embryo on the surface of an agar medium and the germination of an embryo from
a manufactured
seed is weak. This is because inter alia, there are many substantial
differences between bare plant
embryos and manufactured seeds. A key difference is the physical structure of
the seed, which can
have a profound impact on survival of the embryo inside the seed and on
germination success.
There also remain large differences between manufactured seeds and
corresponding
natural seeds. Whereas, the embryo relies on the megagametophyte for nutrients
useful for
germination, the embryo in a manufactured seed relies on the nutritive medium
that is provided in
the manufactured seed.
Also, an agar medium used as a surface on which plant embryos are grown
appears
to have substantially different requirements compared to a nutritive medium
for use in
manufactured seed.
Therefore, there is a need for nutritive media that, when incorporated into
manufactured seeds, provides an improved germination rate compared to
conventional
manufactured seeds.
SUMMARY OF THE INVENTION
In accordance with one aspect of the present invention, manufactured seeds are
provided that comprise a unit of totipotent plant tissue and a nutritive
medium. The nutritive
medium preferably comprises 0 g/L to 30 g/L gel solute, 5 mM to 200 mM carbon
source, 0 mM

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to 20 mM KNO3, 2.5 mM to 5 mM NH4NO3, 0 mM to 0.01 mM KI, 0.01 mM to 20 mM
KH2PO4,
0.5 mM to 5 mM CaC12, 0.1 mM to 10 mM MgSO4, 0 mM to 0.1 mM Na2EDTA, 0 mM to
0.1
mM FeSO4, 0 mM to 0.1 mM MnSO4, 0.01 mM to 1 mM H3BO3, 0.0001 mM to 0.005 mM
ZnSO41 0 mM to 0.0001 mM CoC121 0 mM to 0.0001 mM CuSO4, 0 mM to 0.001 mM
Na2MoO41
0 mM to 0.01 mM thiamine=HCI, 0.001 mM to 0.005 mM pyridoxine=HCI, 0.001 mM to
0.02 mM
nicotinic acid, 0.05 mM to 2.5 mM myo-inositol, and 0.002 mM to 0.2 mM L-
glycine. The
nutritive medium can also include any of various additional components such as
one or more
vitamins, charcoal, smoke suspension, and one or more carbon sources (e.g., a
monosaccharide, a
disaccharide, or a starch).
Any of various different amino acids can be incorporated into the nutritive
medium as
required. Such amino acids can be those that are commonly found incorporated
into proteins
(termed "protein amino acids"). The preferred protein amino acids are one or
more of L-
asparagine, L-glutamine, L-lysine, L-serine, L-proline, L-arginine, L-valine,
L-alanine, L-cysteine,
L-leucine, L-tyrosine, L-threonine, L-phenylalanine, L-histidine, L-glycine, L-
tryptophan, L-
isoleucine, L-methionine, and mixtures thereof.
The nutritive medium can include one or more amino acids that are not commonly
found incorporated into proteins (termed "non-protein amino acids"). There are
over 200 such
amino acids, some of which are argininosuccinate, citrulline, canavanine, and
ornithine. The non-
protein amino acids also encompass the D-stereoisomers of the protein amino
acids.
The nutritive medium can comprise one or more compounds involved in the
metabolism of nitrogen. Representative examples of such compounds include urea
and/or
polyamines such as one or more of spermidine, spermine, and putrescine.
The nutritive medium can include a smoke suspension. A suitable smoke
suspension
contains one or more compounds generated through the process of burning
organic matter such as
cellulosic material.
According to another aspect of the invention, manufactured seeds are provided
that
comprise a modified nutritive medium. Such a medium preferably comprises 0 g/L
to 30 g/L gel
solute, 0 g/L to 5 g/L charcoal, 5 mM to 200 mM carbon source, 0 mM to 30 mM
urea, 0 mM to
20mM KNO3, 2.5 mM to 5 mM NH4NO3, 0 mM to 0.01 mM KI, 0.01 mM to 20 mM KH2PO4,
0.5 mM to 5 mM CaC12, 0.1 mM to 10 MM MgSO41 0 mM to 0.1 mM Na2EDTA, 0 mM to
0.1
mM FeSO41 0 mM to 0.5 mM ferric citrate, 0 mM to 0.1 mM MnSO4, 0 mM to 0.1 mM
MnCI,,
0.01 mM to 1 mM H3BO3, 0.0001 mM to 0.005 mM ZnSO4, 0 mM to 0.0001 mM CoCl2, 0
mM
to 0.0001 mM CuSO4, 0 mM to 0.005 mM CuCl2, 0 mM to 0.001 mM Na2MoO4, 0 mM to
0.001
mM (NH4)2MoO4, 0 mM to 0.01 mM thiamine=HCI, 0 mM to 0.001 mM riboflavin,
0.001 mM to
0.005 mM pyridoxine=HCI, 0 mM to 0.04 mM Ca-pantothenate, 0.001 mM to 0.02 mM
nicotinic
acid, 0 mM to 1.0x10.5 mM biotin, 0 mM to 0.001 mM folic acid, 0.05 mM to 2.5
mM myo-
inositol, 0 mM to 2 mM L-asparagine, 0 mM to 6 mM L-glutamine, 0 mM to 2 mM L-
lysine, 0

CA 02309258 2004-04-13
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mM to 2 mM L-serine, 0 mM to 2 mM L-proline, 0 mM to 8 mM L-arginine, 0 mM to
2 mM Lvaline, 0 mM
to 2 mM L-alanine, 0 mM to 2 mM L-cysteine, 0 mM to 2 mM L-leucine, 0 mM to 2
mM L-tyrosine, 0 mM
to 2 mM L-threonine, 0 mM to 2 mM L-phenylalanine, 0 mM to 2 mM L-histidine,
0.002 mM to 0.2 mM L-
glycine, 0 mM to 2 mM L-tryptophan, 0 mM to 2 mM L-isoleucine, and 0 mM to 2
mM L-methionine. Such
a nutritive medium can also include one or more additional compounds selected
from any of various
vitamins, hormones, a smoke suspension, and sources of carbon. For example,
the modified nutritive
medium can include 0 to 5 g/L charcoal and a carbon source such as a
monosaccharide, a disaccharide, or a
starch.
A modified nutritive medium according to the invention can include one or more
compounds
involved in the metabolism of nitrogen. Representative examples of such
compounds are urea and
polyamines such as spermidine, spermine, and putrescine.
The modified nutritive medium according to the invention can include a "smoke
suspension". A
suitable "smoke suspension" contains one or more compounds that are generated
through the process of
burning organic matter.
The modified nutritive medium can include one or more non-protein amino acids.
According to another aspect of the invention, methods are provided for
germinating a unit of
totipotent plant tissue. A representative embodiment of such a method
comprises the steps of: providing a
nutritive medium as summarized above, providing a unit of totipotent plant
tissue, placing the unit of
totipotent plant tissue in "functional contact" with the nutritive medium, and
placing the manufactured seed
in an environment conducive for plant growth so as to allow the plant tissue
to grow and germinate from the
manufactured seed.
In a first embodiment there is disclosed a manufactured seed comprising: a
structure for receiving a
plant embryo; and a nutritive medium comprising about 5 mM to about 30 mM
urea, about 0.01 mM to
about 8 mM L-arginine, and about 0.001 mM to about 0.01 mM thiamine-HCI.
In a further embodiment the urea concentration is about 5 mM to about 20 mM.
In a further embodiment the arginine concentration is about 0.01 mM to about 4
mM.
In a further embodiment the thiamine-HCI concentration is about 0.001 mM to
about 0.005
mM.
In a further embodiment the nutritive medium further comprises charcoal.
In a further embodiment the nutritive medium further comprises a non-protein
amino acid.
In a further embodiment the non-protein amino acid is ornithine.
In a further embodiment the carbon source comprises sucrose.
In a further embodiment the nutritive medium further comprises a smoke
suspension.
In a further embodiment the nutritive medium further comprises a polyamine
selected from
the group consisting of putrescine, spermidine, spermine, and mixtures
thereof.
In a further embodiment the nutritive medium further comprises at least one
component
selected from the group consisting of about 0.01 mM to 0.1 mM L-glycine, 0.01
mM to 1 mM L-
asparagine, 0.01 mM to 6 mM L-glutamine, 0.01 mM to 1 mM L-lysine, 0.01 mM to
1 mM L-
serine, 0.01 mM to 1 mM L-proline, 0.01 mM to 1 mM L-valine, 0.01 mM to 1 mM L-
alanine, 0.01

CA 02309258 2004-04-13
-4a-
mM to 1 mM L-cysteine, 0.01 mM to 1 mM L-leucine, 0.01 mM to 1 mM L-tyrosine,
0.01 mM to I
mM L-threonine, 0.01 mM to 1 mM L-phenylalanine, 0.01 mM to 1 mM L-histidine,
0.01 mM to
0.1 mM L-glycine, 0.01 mM to 1 mM L-tryptophan, 0.01 mM to 1 mM L-isoleucine
and 0.01 mM
to 1 mM L-methionine.
In a further embodiment the plant embryo is a gymnosperm plant embryo.
In a further embodiment the gymnosperm is Pine.
In a further embodiment there is disclosed a method for germinating a plant
embryo,
comprising placing the plant embryo in functional contact with a nutritive
medium comprising
about 5 mM to about 30 mM urea, about 0.01 mM to about 8 mM L-arginine, and
about 0.001 mM
to about 0.01 mM thiamine-HC1 in a manufactured seed.
In a further embodiment the nutritive medium further comprises at least one
component selected
from the group consisting of about 0.01 mM to 0.1 mM L-glycine, 0.01 mM to 1
mM L-asparagine, 0.01
mM to 6 mM L-glutamine, 0.01 mM to 1 mM L-lysine, 0.01 mM to 1 mM L-serine,
0.01 mM to 1 mM L-
proline, 0.01 mM to 1 mM L-valine, 0.01 mM to 1 mM L-alanine, 0.01 mm to 1 mM
L-cysteine, 0.01 mM
to 1 mM L-leucine, 0.01 mM to 1 mM L-tyrosine, 0.01 mM to 1 mM L-threonine,
0.01 mm to 1 mm L-
phenylalanine, 0.01 mM to 1 mM L-histidine, 0.01 mM to 0.1 mM L-glycine, 0.01
mM to 1 mM L-
tryptophan, 0.01 mM to 1 mM L-isoleucine and 0.01 mM to 1 mM L-methionine.
In a further embodiment the plant embryo is a gymnosperm plant embryo.
In a further embodiment the gymnosperm is Pine.
In a further embodiment there is disclosed a method for germinating a plant
embryo comprising
placing the plant embryo in a manufactured seed according to any one of the
other embodiments.
The foregoing and additional features and advantages of the invention will be
more readily apparent
from the following detailed description.
DETAILED DESCRIPTION
1. Basic Nutritive Medium
A basic nutritive medium according to the invention can have a profile within
one of the two ranges
provided below in Table 1. The column labeled "Concentration Range 1 "pertains
to preferred concentration
ranges of the representative components. The column labeled "Concentration
Range 2"pertains to more
preferred concentration ranges.
Table 1
Basic Medium
Medium Component Concentration Ranee 1 Concentration Range 2
gel solute 0 g/L to 30 g/L 10 g/L to 25 g/L
carbon source 5 mM to 200 mM 25 mM to 175 mM
KNO3 0mMto20mM 10mmto15mm
NH4NO3 2.5 mM to 5 mM 2 mM to 4 mM
KI 0 mm to 0. 01 mm 0.002 mM to 0. 005 mM

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Medium Component Concentration Range 1 Concentration Range 2
KH2PO4 0.01 mM to 20 mM 0.05 mM to 18 mM
CaC12 0.5 mM to 5 mM ImMto3mM
MgSO4 0.1 mm to lO mm 0.2 mM to 3 mM
Na2EDTA 0 mm to 0.1 mm 0.01 mM to 0.8 mM
FeSO4 0 mm to 0.1 mm 0.01 mM to 0.05 mM
MnSO4 0 mm to 0.1 mm 0.02 mM to 0.5 mM
H3BO3 0.01 mm to 1 mM 0.1 mM to 0.5 mM
ZnSO4 0.0001 mM to 0.005 mM 0.0003 mM to 0.004 mM
CoC12 0 mm to 1x104 mm 1x10-5 mM to 1x104 mM
CuSO4 0 mm to 1x10' mm 1x10.5 mm to 1x104 mM
Na2MoO4 0 mm to 0.001 mm 1x104 mM to 0.001 mM
thiamine=HCI 0 mm to 0.01 mm 0.001 mM to 0.005 mM
pyridoxine-HCI 0.001 mM to 0.005 mM 0.001 mM to 0.004 mM
nicotinic acid 0.001 mM to 0.02 mM 0.001 mm to 0.01 mm
myo-inositol 0.05 mM to 2.5 mM 0.1 mm to 1 mm
L 1 cine 0.002 mM to 0.2 mM 0.01 mm to 0.1 mm
In Table 1, concentrations given in g/L denote grams of the respective
component per
liter of medium. The basic medium preferably contains at least fifteen of the
components listed in
Table 1 and more preferably at least twenty of the components listed in Table
1.
The basic nutritive medium can be sterilized by autoclaving, filter
sterilizing, or by
using another suitable method. The nutritive medium can be incorporated into
manufactured seed
as described in, e.g., U.S. Patent No. 5,701,699 to Carlson.
The basic nutritive medium facilitates germination of seedlings from
manufactured seed containing
such medium.
In Table 1, certain components (e.g., thiamine) are listed as the HCI form.
One of
ordinary skill in the art will appreciate that the basic nutritive medium can
be formulated using such
components that do not contain HCI. For example, it is possible to use
thiamine without the HCI
instead of thiamine=HCI.
The present invention also encompasses nutritive media, derived from the basic
nutritive medium described above, having an altered profile of components,
including one or more
added components.
Such additional components include one or more protein amino acids, one or
more
non-protein amino acids, charcoal, smoke suspension, one or more carbon
sources, and any of
various components of the metabolic pathways pertaining to nitrogen
utilization by plants.
Components of these pathways include, but are not limited to, urea,
putrescine, spermidine, and
spermine.

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II. Additional Components
a. Amino Acids
Representative protein amino acids that can be added to the basic nutritive
medium
include one or more of the following: L-glutamine, L-arginine, L-proline, L-
asparagine, L-lysine,
L-serine, L-valine, L-alanine, L-cysteine, L-leucine, L-tyrosine, L-threonine,
L-phenylalanine, L-
histidine, L-tryptophan, L- glycine, and L-isoleucine. A preferred
concentration of protein amino
acids in the nutritive medium is 0 mM to 8 mM, more preferably 0.01 mm to 4
mM.
In addition to, or instead of, the protein amino acids listed above, the
nutritive
medium may comprise one or more amino acids that are not usually constituents
of proteins. Such
"non-protein" amino acids are commonly found in certain plants as storage
proteins, and over 200
such non-protein amino acids are known. (As used herein, the "non-protein"
amino acids include
any of the D-stereoisomers of the protein amino acids.) By way of example,
omithine, canavanine,
or other non-protein amino acid (or mixture thereof) can be added to the
nutritive medium at a
concentration of 0 mM to 3 mM, more preferably 0.1mM to 1.25 mM per non-
protein amino acid.
b. Charcoal
According to the invention charcoal can favorably influence germination when
added
to a nutritive medium. Preferably, the charcoal is in the form of a powder and
is activated by
pretreatment with HC1. Charcoal appears to facilitate increased hypocotyl
length and increased
radical length of germinating plant embryos of certain species of plants. In
such instances the
preferred concentration of charcoal is 0 g/L to 5 g/L, more preferably 2 g/L
to 3 g/L.
c. Smoke Suspension
The by-products of burning organic matter can contribute to increased
germination.
Solutions containing these by-products can be generated by burning organic
matter (e.g., wood or
other cellulosic material), washing the charred material with water, and
collecting the water.
Solutions can also be obtained by heating the organic matter (e.g., wood or
other cellulosic
material), and condensing and diluting volatile substances released from such
heating.
Certain types of smoke suspensions can be purchased from commercial suppliers.
For example, and not intending to be limiting, Wright's Concentrated Hickory
Seasoning Liquid
Smoke may be purchased from B&G foods, Inc. Roseland, NJ 07068.
A nutritive medium according to the invention comprising a smoke suspension
can
increase organ length and improve germination of certain plant species. Smoke
suspension can be
incorporated into the nutritive medium in any of various forms. For instance,
smoke suspension
can be incorporated as an aerosol, a powder, or as activated clay. The
preferred concentration of
Wright's Concentrated Hickory Seasoning Liquid Smoke liquid smoke suspension,
if present, is

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between 0.0001 mL smoke suspension per liter of medium to I mL smoke
suspension per liter of
medium.
d. Carbon Source
In the germination of certain plant embryos (e.g., maize, sorghum, pearl
millet),
hydrolysis of sucrose to glucose and fructose (as representative carbon
sources) precedes uptake of
the carbon sources. Embryos of other crop plants appear to take up sucrose
prior to hydrolysis.
Therefore, depending on the species being germinated from the manufactured
seed, the carbon
source in a nutritive medium according to the invention can be, for example,
one or more
monosaccharides (as representative "simple" carbon sources). Alternatively or
in addition to one
or more simple carbon sources, the nutritive medium can include one or more
"complex" carbon
sources such as any of various disaccharides (e.g., sucrose), and any of
various polysaccharides
(e.g., starch). A preferred concentration of the carbon source (either as a
single compound or as a
mixture of compounds) in the medium is 5 mM to 200 mM, more preferably 25 MM
to 175 mM.
e. Components of Pathways Involved in Nitrogen
Utilization
A growing plant needs to be able to take in nitrogen and metabolically utilize
it. A
representative source of nitrogen is arginine. Therefore, certain compounds
involved in the
synthesis and degradation of arginine can be especially useful as additives to
a nutritive medium
according to the invention. One such compound is urea. A preferred
concentration of urea is 0
mM to 30 mM. A more preferred concentration of urea, if used, is 5 mM to 20
mM.
In addition to or as an alternative to urea, any of various polyamines
involved in the
degradation of arginine can be added to a nutritive medium according to the
invention. Suitable
polyamines can be selected from the breakdown products of certain amino acids.
By way of
example, a preferred concentration of spermidine, putrescine, and/or
spermidine is 0 mM to 0.5
mM per compound. A more preferred concentration is 0.01 mM to 0.5 mM per such
compound
present in the medium.
M. Modified Nutritive Medium
To facilitate further increases in germination success rate of (or a higher
percentage
of normal germinants from) manufactured seeds containing certain species of
plant embryos and a
nutritive medium according to the invention, the nutritive medium can comprise
any of various
additional components.
Representative modified nutritive media according to the invention can have a
profile
within one of the two ranges provided in Table 2, below. In Table 2, the
column labeled
"Concentration Range 1" pertains to preferred concentration ranges of the
respective components.
The column labeled "Concentration Range 2" pertains to more preferred
concentration ranges.

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Table 2
Modified Medium
Medium Component Concentration Range 1 Concentration Range 2
gel solute 0 g/L to 30 g/L 10 g/L to 25 g/L
Charcoal 0 g/L to 5 g/L 2 g/L to 3 g/L
carbon source 5 mM to 200 mM 25 mM to 175 mM
Urea O mM to 30 mM 5 mM to 20 mM
KNO3 0 mM to 20 mM 10 mM to 15 mM
NH4NO3 2.5 mM to 5 mM 2 mM to 4 mM
KI 0 mm to 0.01 mm 0.002 mM to 0.005 mM
KH2POa 0.01 mM to 20 mM 0.05 mM to 18 mM
CaCl2 0.5mMto5mM 1mMto3mM
MgSO4 0.1 mm to 10 mm 0.2 mM to 3 mM
Na2EDTA 0 mm too. I mm 0.01 mM to 0.8 mM
FeSO4 0 mm too. I mm 0.01 mM to 0.05 mM
ferric citrate 0 mM to 0.5 mM 0.1 mM to 0.4 mM
MnSO4 0 mm to 0.1 mm 0.02 mM to 0.5 mM
MnCl2 0 mm too. I mm 0.01 mM to 0.05 mM
H3BO3 0.01 mm to 1 mm 0.1 mM to 0.5 mM
ZnSO4 0.0001 mM to 0.005 mM 0.0003 mM to 0.004 mM
CoC12 0 mm to 1x10'4 mm 1x10-5 mM to 1x10-4 mM
CuC12 0 mM to 0.005 mM 0.001 mM to 0.005 mM
CuSO4 0 mM to Ix1O4 mM 1x105 mM to 1x104 mM
Na2MoO4 0 mm to 0.001 mm Ix1O4 mM to 0.001 mM
(NH4)2MoO4 0 mm to 0.001 mm 1x10-4 mM to 0.001 mM
thiamine=HCI 0 mm to 0.01 mm 0.001 mM to 0.005 mM
Riboflavin 0 mm to 0.001 mm 1x10'4 mM to 0.001 mM
pyridoxine=HCl 0.001 mM to 0.005 mM 0.001 mM to 0.004 mM
Ca-pantothenate 0 mM to 0.04 mM 0.0005 mM to 0.015 mM
nicotinic acid 0.001 mM to 0.02 mM 0.001 mm to 0.01 mm
Biotin 0 mm to 1.0x 10-5 mm 1x107 mM to 1x105 mM
folic acid 0 mm to 0.001 mm 1x104 mM to 1x103 mM
myo-inositol 0.05 mM to 2.5 mM 0.1 mm to 1 mm
L-asparagine 0 mM to 2 mM 0.01 mm to 1 mm
L-glutamine 0 mM to 6 mM 0.01 mM to 3 mM
L-lysine 0 mM to 2 mM 0.01 mm to 1 mm
L-serine 0 mM to 2 mM 0.01 mm to 1 mm
L-proline 0 mM to 2 mM 0.01 mm to 1 mm
L-arginine 0 mM to 8 mM 0.01 mM to 4 mM
L-valine 0 mM to 2 mM 0.01 mm to 1 mm
L-alanine 0 mM to 2 mM 0.01 mm to 1 mm
L-cysteine 0 mM to 2 mM 0.01 mm to 1 mm
L-leucine 0 mM to 2 mM 0.01 mm to 1 mm
L-tyrosine 0 mM to 2 mM 0.01 mm to 1 mm
L-threonine 0 mM to 2 mM 0.01 mm to 1 mm
L-phenylalanine 0 mM to 2 mM 0.01 mm to 1 mm
L-histidine 0 mM to 2 mM 0.01 mm to 1 mm
L-glycine 0.002 mM to 0.2 mM 0.01 mm too. I mm
L-tryptophan 0 mM to 2 mM 0.01 mm to 1 mm
L-isoleucine 0 mM to 2 mM 0.01 mm to 1 mm
L-methionine 0 mM to 2 mM 0.01 mm to 1 mm
non-protein amino acid 0 mM to 3 mM 0.01 mM to 1.25 mM

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Medium Component Concentration Range 1 Concentration Range 2
smoke suspension 0 to 10 ml/L medium 0.1 mL/L to 1 mL/L medium
Polyamine 0 mM to 0.5 mM 0.01 mM to 0.5 mM
In Table 2, concentrations given in g/L denote grams of the respective
component per liter
of medium. The modified medium preferably contains at least one of the protein
amino acids listed
in Table 2, and more preferably at least five of the protein amino acids
listed in Table 2. In
addition to the protein amino acids, the modified medium contains at least 15
of the other
components (that are not protein amino acids) listed in Table 2, and more
preferably at least 20 of
the other components listed in Table 2.
In Table 2, a "polyamine" is any of various compounds comprising molecules
having two
or more amine groups, such as, but not limited to, putrescine, spermine, and
spermidine.
The concentrations of individual components can be varied depending upon the
species of plant embryo contained in the manufactured seed and upon the
environment in which the
seed is to be germinated.
IV. Plant Tissue
Plant tissue incorporated into a manufactured seed comprising a nutritive
medium
according to the invention is preferably totipotent. As used herein,
"totipotent" refers to a capacity
to grow and develop into a normal plant. Totipotent plant tissue has both the
complete genetic
information of a plant and the ready capacity to develop into a complete plant
if cultured under
favorable conditions. Totipotent plant tissue is obtainable from several areas
of a plant, such as
meristematic tissue and plant embryonic tissue.
Meristematic tissue is comprised of undifferentiated plant cells that divide
to yield
other meristematic cells as well as differentiated cells that elongate and
further specialize to form
structural tissues and organs of the plant. Meristematic tissue is located,
for example, at the
extreme tips of growing shoots or roots, in buds, and in the cambium layer of
woody plants.
Plant embryonic tissue can be found (in the form of a "zygotic" embryo) inside
a
botanic seed produced by sexual reproduction. Also, plant "somatic" embryos
can be produced by
culturing totipotent plant cells such as meristematic tissue under laboratory
conditions in which the
cells comprising the tissue are separated from one another and urged to
develop into minute
complete embryos. Alternatively, a process termed "cleavage polyembryony"
known in the art can
be induced during natural embryo development in seed. For simplicity,
totipotent plant tissue is
referred to herein simply as the "embryo", unless stated otherwise.

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V. Gel Solute
A nutritive medium according to the invention can include a substance that
causes the
medium to be a semisolid or have a congealed consistency under normal
environmental condition
(see Definitions below). A detailed description of various types of gel
solutes can be found in U.S.
Patent No. 5,564,224 to Carlson.
VI. Oxygen-Absorbing Substances
U.S. Patent No. 5,564,224 discloses various
oxygen-absorbing substances (also termed "oxygen-carrying" substances). Such
substances can be
added to a nutritive medium according to the present invention to enhance both
the absorption of
oxygen and the retention of oxygen by the nutritive medium, thereby allowing
the medium to
maintain a concentration of oxygen that is higher than would otherwise be
present in the medium
solely from the absorption of oxygen from the atmosphere.
The amount and type of oxygen-carrying substance that is added to the medium
will
vary depending on the species being germinated from the manufactured seed. In
some cases a
nutritive medium including an oxygen-carrying substance does not facilitate
improved germination
compared to an otherwise identical nutritive medium lacking an oxygen-carrying
substance.
VII. Preferred Uses of Nutritive Medium
2 0 Nutritive media according to the invention are useful for manufacturing
and
germinating manufactured seeds in a variety of different contexts. As
previously mentioned, the
optimal concentrations of the various components can be adjusted within the
respective stated
ranges depending upon the species of embryo and the conditions under which the
manufactured
seed will be sown.
Representative methods used for making manufactured seeds including a
nutritive
medium are described in U.S. Patent No. 5,701,699 to Carlson.
VIII. Definitions
The following terms are defined as follows:
"Unit of totipotent plant tissue" refers to the minimum mass of plant tissue
that can
give rise to a mature plant. For example, a single cell can mature into a
plant, and is therefore, a
unit of totipotent plant tissue. However, in a practical sense, a suitable
unit of totipotent plant
tissue comprises multiple cells (hence the term "tissue") such as in a plant
embryo.
"Functional contact" is any contact of the plant tissue with the nutritive
medium
that allows for the flow of water and components from the nutritive medium to
the unit of totipotent
plant tissue. Therefore, functional contact can include actual physical
contact or indirect contact

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resulting from, e.g., enclosing the plant tissue with a cotyledon restraint
such as described in U.S.
Patent No. 5,687,504,
"Non-protein amino acid" is any amino acid that is not normally found as a
constituent of proteins, for example, but not limited to, argininosuccinate,
citrulline, homoserine,
and ornithine.
"Gel solute" is a solute that, when added to the medium, causes the medium to
become a semisolid or otherwise congeal. A preferred gel solute is neither
cytotoxic nor
substantially phytotoxic. Candidate gel solutes include, but are not limited
to, the following:
sodium alginate, agar, agarose, amylose, pectin, dextran, gelatin, starch,
amylopectin, modified
celluloses such as methylcellulose and hydroxyethylcellulose and
polyacrylamide.
"Somatic embryo" is a plant embryo produced via the laboratory culturing of
totipotent plant cells or by induced cleavage polyembryony.
"Zygotic embryo" is a plant embryo removed from a natural botanic seed of the
corresponding plant.
"Germinant" is a plant embryo that has undergone sufficient growth and
development to protrude from a manufactured seed, analogous to a plant embryo
protruding from a
natural botanic seed.
"Radicle" is that part of a plant embryo that develops into the primary root
of the
resulting plant.
"Cotyledon" refers generally to the first, first pair, or first whorl
(depending on the
plant type) of leaf-like structures on the plant embryo that function
primarily to make food
compounds in the seed available to the developing embryo, but in some cases
act as food storage or
photosynthetic structures.
"Hypocotyl" is that portion of a plant embryo or seedling located below the
cotyledons but above the radicle.
"Epicotyl" is the portion of the seedling stem that is above the cotyledons.
"Hypocotyl length" pertains to the length of the hypocotyl at the time the
hypocotyl
was measured.
"Radicle length" pertains to the length of the radicle at the time the radicle
was
measured.
"Normalcy" denotes the presence of all expected parts of a plant (e.g.,
radicle,
hypocotyl, cotyledon(s), epicotyl) at time of evaluation. In the case of
gymnosperms, normalcy is
characterized by the radicle having a length greater than 3 mm and no visibly
discernable
malformations compared to the appearance of control bare embryos grown on the
surface of
nutrient agar or the like.
It is important to note that, as long as all parts of an embryo have
germinated, the
corresponding germinant probably has the potential to become a normal
seedling. There is no

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reason to believe that any malformations observed in the following examples
below are fatal to
germinants. Noting the quantity and quality of malformation is a convenient
way to comparatively
evaluate the various methods and means employed for making manufactured seeds.
Fortunately,
plant embryonic tissue is exquisitely sensitive to non-natural conditions and
manifests that
sensitivity in ways discernable to a trained observer.
Example 1
Preparation of the Nutritive Medium
The basic nutritive medium and the modified nutritive medium are preferably
prepared from pre-made stocks. The required amount of each stock solution
(that is not heat-labile)
is added to water. Non-stock chemicals (such as sucrose, charcoal, and agar)
can be weighed out
and added directly to the solution. After all the non-heat-labile chemicals
and compounds are
added, the medium is brought up to an appropriate volume and the pH is
adjusted. The medium is
then sterilized, e.g., by autoclaving. Pre-sterilized (e.g., by filtration)
heat-labile components are
added after the medium is removed from the autoclave and has cooled. The
medium can
additionally contain one or more antibiotics and/or antimycotics. For example,
the medium can
contain 0.1 mg/L GA4n (available from Abbot Laboratories, North Chicago, IL)
and/or 1 mL/L
media Sigma product # A7292. Both of these products contain an antibiotic and
an antimycotic.
Additionally, since both products are heat-labile, they are preferably added
after the medium has
cooled to below 40 C.
The medium used in the following examples contained the components listed in
Table
3. These particular concentrations were chosen for the purposes of consistency
from one example
to the next and are not intended to be limiting in any way. The medium chosen
could have been
any of various nutritive media within the scope of Table 1 or Table 2.
Table 3
Specific Media Used for Example Purposes
Medium Component Modified Medium Basic Medium
Charcoal 2.5 g/L 2.5 g/L
PH 5.7 5.7
Sucrose 146.07 mM 58.43 g/L
Agar 18 g/L 18 g/L
Urea 13.32 mM 0
KNO3 0 11.57 mM
NH4N03 3.74 mM 2.577 mM
KI 0 0.0025 mM
KH,PO4 13.23 mM 0.625 mM
CaCI2 1.54 mM 1.133 mM
MgSO4 1.98 mM 0.367 mM
Na2EDTA 0 0.05 mM
FeSO4 0 0.027 mM
ferric citrate 0.245 mM 0
MnSO 0 0.045 mM

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Medium Component Modified Medium Basic Medium
MnC12 0.019 mm 0
H3B03 0.162 mM 0.05 mM
ZnSO4 0 8.4xl0-3 mM
CuC12 0.0023 mM 0
CuSO4 3.2x10-5 mM
CoC12 0 2.9x10-5 mM
Na2MoO4 0 4.4x104 mM
(NH4)2MoO4 0.0003 mM 0
GA4n 0.1 mg/L 0.1 mg/L
thiamine=HCI 0.003 mM 0.003 mM
Riboflavin 0.0003 mM 0
Pyridoxine-HCl 0.0012 mM 0.0024 mM
Ca-pantothenate 0.001 mM 0
nicotinic acid 0.0081 mM 0.0041 mM
Biotin 4.09x10-6 mM 0
folic acid 0.0003 mM 0
myo-inositol 0.555 mM 0.555 mM
L-asparagine 0.8076 mM 0
L-glutamine 1.8248 mM 0
L-lysine 0.1624 mM 0
L-serine 0.7613 mM 0
L-proline 0.463 mM 0
L-arginine 1.0471 mM 0
L-valine 0.455 mM 0
L-alanine 0.5983 mM 0
L-cysteine 0.2204 mM 0
L-leucine 0.6099 mM 0
L-tyrosine 0.2942 mM 0
L-threonine 0.2241 mM 0
L-phenylalanine 0.3227 mM 0
L-histidine 0.1721 mM 0
L-glycine 0.71 mM 0.027 mM
L-tryptophan 0.1307 mM 0
L-isoleucine 0.2036 mM 0
L-ornithine 0 0
L-methionine 0.1789 mM 0
Pluronic F-68 3.6 g/L 3.6 g/L
DC-200 silicone oil 240 mL/L 240 mL/L
Generally, the sterilization of different components can be performed using
any of various
suitable techniques that can be performed at various stages in preparation.
For example, a stock
solution can be made that contains one or more of the components in the media.
This stock
solution can be sterilized before addition to the medium, or after addition to
the medium. The
solutions can be sterilized by filtration, exposure to UV light, autoclaving,
or any other suitable
means.
The sterilization principles described above can also be applied to other
media additives.
For example, amino acids, carbon sources, smoke suspension, or polyamines can
be made into a

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stock solution that is filter sterilized. The resulting sterilized stock
solutions can then be added to
the medium after an intermediate solution containing other components has been
autoclaved. On
the other hand, these additives might be added directly to the medium and the
medium as a whole
subsequently sterilized.
Example 2
General Description of the Process of Making Manufactured Seeds
Representative methods used for making manufactured seeds are described in
U.S. Patent
Nos. 5,701,699 and 5,427,593 to Carlson.
After the manufactured seeds are prepared they can be sown in any of a variety
of
environments, such as in sand, vermiculite, sterile soil, and/or in the field
(natural soil). For
purposes of consistency the following examples contain data that were
generated from the growth of
manufactured seeds in sterile sand. For example, Cole "` sand which is
available from a variety of
gardening supply stores can be used.
A suitable amount of sterile sand can be prepared by baking 2 L of sand at a
temperature
of 375 F for 24 hours. The sand is then added to pre-sterilized trays and 285
mL water is added.
Furrows are then formed and the box is sealed. The box containing the sand is
then autoclaved for
1 hour at 121 C and I atm pressure.
The manufactured seeds are then sown in the sand and allowed to germinate.
Typically,
2 0 the manufactured seeds are cultured under continuous light at room
temperature (23 C) for four to
five weeks.
Example 3
Addition of Charcoal to the Nutritive Medium
This example is directed to the effect of adding charcoal to the basic
nutritive medium.
Douglas-fir zygotic embryos were used in manufactured seed containing the
basic medium. The
control group included 36 manufactured seeds, and the test group included 36
manufactured seeds.
Seeds of the test group contained the basic nutritive medium with the addition
of 2.5 g/L charcoal.
Seeds of the control group contained the basic nutritive medium without
charcoal.
All seeds were germinated in sterile sand as described above. The measurements
were
3 0 taken 25 days after the seeds were sown. The results indicate that
charcoal can have a significant
beneficial effect on both hypocotyl and radical length (see Table 4). The
statistical differences
were calculated using the SAS program PROC GLM (general linear models),
available from
Statistical Analysis Systems, Inc. Cary North Carolina, USA.

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Table 4
Average Seedling Dimensions Produced from Each Treatment
Normalcy Radical Length Hypocotyle Cotyledon
Length Length
Basic Medium 58.33% 0.5667 cm 2.2222 cm 1.24118 cm
Basic Medium + 86.11 % 0.9333 cm 2.7500 cm 1.22857 cm
Charcoal
*Significant differences at a = 0.05 were detected using the PROC GLM program.
However, no significant differences in cotyledon length were observed between
the
test and control group. Additionally, manufactured seeds of the test group
showed a significant
improvement in normalcy rates.
Example 4
The Effect of Adding Amino Acids and Polyamines to the Nutritive Medium
In this example the addition of amino acids to the basic nutritive medium
described in
Table 3 was evaluated to determine the effect of the added amino acids on the
germination of
manufactured seeds containing such nutritive medium. The manufactured seeds
were constructed
by the methods described in Example 2. The control group consisted of
manufactured seeds
containing the basic medium. Test groups consisted of manufactured seeds
containing the basic
nutritive medium additionally comprising 0.50 mM L-glutamine, 0.125 mM L-
proline, and 0.125
mM L-asparagine plus one additional test component (a different test component
for each test
group). The test components were 0.50 mM L-arginine, 0.50 mM ornithine, or a
combination of
polyamines (0.10 mM putrescine, 0.10 mM spermidine, and 0.10 mM spermine).
The manufactured seeds of all treatments (control and test groups) contained
Douglas fir
embryos. There were six replicates with six manufactured seeds from each
treatment in each
replicate. The seeds were germinated in sterile sand as described in Example
2. The seeds were
allowed to germinate for 25 days after which organ length and normalcy of
germinants were
assessed. These results are shown in Table 5.
Table 5
Average Seedling Dimensions Produced from Each Treatment
Treatment Normalcy % Radical Hypocotyl Cotyledon
Length Length Len h
1. Standard Medium 65.87% 0.67 cm 2.20 cm 1.42 cm
2. Standard Medium + 77.8 % 1.19 cm 2.71 cm 1.53 cm
2.5 g/L charcoal + 0.50
mM L-glutamine, 0.125
mM L-proline, 0.125 mM
L-asparagine, 0.50 mM L-
ar inine
3. Standard Medium + 83.3 % 1.02 cm 2.51 cm 1.39 cm
2.5 g/L charcoal + 0.50

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mM L-glutamine, 0.125
mM L-proline, 0.125 mM
L-asparagine, 0.50 mM
ornithine
4. Standard Medium + 52.2 % .88 cm 2.34 cm 1.21 cm
2.5 g/L charcoal + 0.50
mM L-glutamine, 0.125
mM L-proline, 0.125 mM
L-asparagine, 0.10 mM
putrescine, 0.10 mM
spermine and 0.10 mM
s rmidine
*Significant differences at a = 0.05 were detected using the PROC GLM program.
The results show that the largest difference between the test groups and the
control was in
radical length, and the least effect was found in cotyledon length.
Example 5
Comparison of Loblolly Pine Zygotic Embryos Germinated From Manufactured Seeds
Containing Basic Medium versus the Modified Medium
In this example the basic medium, without pluronic F-68 and DC-200 silicone
oil,
described in Table 3 was supplemented to bring the sucrose concentration to 50
g/L. This
supplemented basic medium was then compared to the modified medium described
in Table 3. The
manufactured seeds were constructed containing loblolly pine embryos according
to Example 1.
The study was designed as a randomized complete block. There were six blocks.
Each
block consisted of a germination box as described above with ten manufactured
seeds of each
treatment sown therein. The seeds were allowed to germinate for 34 days in
sterile sand, after
which the seeds were removed from the sand and the normalcy, presence or
absence of an epicotyl,
radical, hypocotyl, and cotyledon lengths of the resulting germinants were
measured. The data is
presented in Table 6.
Table 6
Average Seedling Dimensions Produced from Each Treatment
Treatment Normalcy E ip cotyl Radical Hypocotyl Cotyledon
Presence Length Length Length
Modified Medium (Table 3) 53.3 % 21.7 % 0.97 cm 2.34 cm 1.58 cm
Basic Medium (Table 3)+
0 /L sucrose 16.7 % 3.3 % 0.69 cm 1.82 cm 0.91 cm
Significant differences at a = 0.05 were detected using the PROC GLM program.
Many differences in growth and germination were observed with manufactured
seeds
containing the modified nutritive medium versus manufactured seeds containing
the basic nutritive
medium. Manufactured seeds containing the modified nutritive medium exhibited
significantly
SUBSTITUTE SHEET (RULE 26)

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better germination, normalcy, epicotyl presence, and radical elongation than
manufactured seeds
containing the basic nutritive medium.
Example 6
Comparison Douglas Fir Zygotic Embryos Germinated From Manufactured Seeds
Containing
Basic Medium versus the Modified Medium
In this example the two media formulations listed in Table 3 were incorporated
into
respective populations of manufactured seeds containing Douglas fir zygotic
embryos. This was
done to determine which of the two media was more effective at producing
normal germination and
organ elongation of such embryos from manufactured seeds. A third treatment
was included to
determine the effect of sucrose concentration on germination of such embryos.
Manufactured seeds
were constructed as described in Example 1. The study consisted of six
replicate germination
boxes with ten manufactured seeds/treatment/box. The seeds were allowed to
germinate in sterile
sand as described above for 27 days. Measurements were then taken and
recorded. The data are
presented in Table 7.
Table 7
Average Seedling Dimensions Produced from Each Treatment
Treatment Normalcy Radical Hypocotyl Cotyledon
Length Length Length
1) Medium 1 90.0 % 0.95 cm 2.94 cm 1.26 cm
2) Medium 2 with 20 g/L 80.0 % 1.20 cm 3.52 cm 1.61 cm
sucrose
3) Medium 2 with 50 g/L 78.3 % 1.83 cm 3.82 cm 1.48 cm
sucrose
Significant differences at a = 0.05 were detected using the PROC GLM program.
These data indicate that the modified medium with either concentration of
sucrose
significantly increased organ elongation of Douglas fir germinants from
manufactured seed. In
comparison to the basic medium, the modified medium with 50 g/L sucrose
produced the longest
radicals and hypocotyls and increased cotyledon length.
Example 7
The Addition of Liquid Smoke to Nutritive Medium
This example is directed to the effect of a nutritive medium including "liquid
smoke" (as a
representative smoke suspension) on the germination of manufactured seeds
containing such a
medium. The starting medium used was the modified medium listed in Table 3
without Pluronic F-
68 and DC-200 silicone oil. To this starting medium various amounts of
Wright's Concentrated
Hickory Seasoning Liquid Smoke were added. (Wright's Concentrated Hickory
Seasoning Liquid

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Smoke is available from B&G foods, Inc. Roseland, NJ 07068). Treatment groups
contained either
0.01 L, 0.1 L, 1 ML, 10.tL, 100 L, I mL, or 10 mL of liquid smoke per 100
mL medium.
The manufactured seeds were prepared as described in Example 2, using Loblolly
pine
somatic embryos. The seeds were sown in germination boxes containing sterile
sand as described
above. The study consisted of six germination boxes and eight (30 seeds each)
treatments with five
seeds per treatment per box (a total of 240 manufactured seeds). After 34 days
the seeds were
scored for normalcy and organ elongation. Results are shown in Table 8.
Table 8
Average Seedling Dimensions Produced from Each Treatment
Milliliters of Percent Root Length Hypocotyl Cotyledon % With
Liquid Smoke in Normal Length Lenzth E ip cotyl
100 mL medium
Ti: 0.01 mL 47 0.93 cm 2.43 cm 1.70 cm 30
(control)
T2: 0.01 L 50 1.43 cm 2.32 cm 1.74 cm 43
T3: 0.1 L 63 1.45 cm 2.47 cm 1.72 cm 53
T4: 1.0 L 77 1.31 cm 2.34 cm 1.77 cm 53
T5: 10 L 40 0.91 cm 2.38 cm 1.65 cm 30
T6: 0.1 mL 53 1.24 cm 2.42 cm 1.60 cm 27
T7: 1.0 mL 23 0.81 cm 2.00 cm 1.53 cm 27
T8: 10.0 mL 0 unchanged unchanged unchanged 0
Significant Block No No No No No
Difference
Significant differences at a = 0.05 were detected using the PROC GLM program.
The treatment group in which the nutritive medium contained 1.0 L liquid
smoke per 100
mL medium yielded the greatest response based on normalcy score (77%).
Treatment with either
0.1 L or 0.01 L liquid smoke per 100 mL medium yielded significantly longer
root length, and
treatment with 0.01 ML, 0.1 L, or 1 L of liquid smoke per 100 mL medium
yielded significantly
higher percentage of germinants having an epicotyl compared to the treatment
with 10.0 L of
liquid smoke per 100 mL medium. Though not significant, the treatments with
0.1 L and 1.0 L
of Liquid Smoke per 100 mL medium yielded 43% more germinants having an
epicotyl than the
control in which the nutritive medium did not contain Liquid Smoke.
Example 8
Effects of a Carbon Source on Germination of Manufactured Seeds
2 5 In this example several carbon sources were tested at various
concentrations in the basic
medium described in Table 3. Manufactured seeds were prepared as described in
Example 1.
Media formulations were based on the basic medium except that the following
carbon sources and
concentrations were used: Treatment 1 - basic medium containing 20 g/L
sucrose; Treatment 2 -
basic medium containing 20 g/L maltose; Treatment 3 - basic medium containing
20 g/L glucose;

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Treatment 4 - basic medium containing 10 g/L maltose; Treatment 5 - basic
medium containing 10
g/L glucose, and Treatment 6 - basic medium containing 20 g/L honey. Stock
solutions of the
glucose and maltose used to prepare media used in Treatments 2-5 were filter
sterilized instead of
autoclaved. In this study there were six replicate germination boxes
containing sterile sand with
five manufactured seeds per Treatment in each box.
The results of this study showed no significant differences in normalcy among
the
various Treatments using SAS data analysis software (p = 0.0772). However,
more normal
germinants were produced from manufactured seeds containing a higher
concentration of any of the
carbon sources than from manufactured seeds containing the lower concentration
of the carbon
sources (with the exception of honey). Significant differences were also
detected in radical length
p = 0.0001). The LSD mean separation procedure (run using the SAS data
analysis software)
revealed that Treatment 2 (20 g/L glucose) produced radicals significantly
longer than all other
Treatments except the control (Treatment 1). Ranking the treatments in order
of longest to shortest
radicals yielded the following: Treatment 3 produced 0.85 cm radicals;
Treatment I produced 0.68
cm radicals; Treatment 6 produced 0.61 cm radicals; Treatment 5 produced 0.43
cm radicals;
Treatment 2 produced 0.38 cm radicals; and Treatment 5 produced 0.34 cm
radicals.
Example 9
Effects of Sucrose Concentration on Germination of Manufactured Seeds
Containing Loblolly
Pine Zygotic Embryos
In this Example various concentrations of sucrose were incorporated into the
basic medium
used in manufactured seeds containing loblolly pine zygotic embryos.
Manufactured seeds were
produced as described in Example 1. Treatment groups consisted of manufactured
seeds containing
the basic medium as described in Table 3 with either 40 g/L filter-sterilized
sucrose or 50 g/L
filter-sterilized sucrose. The manufactured seeds were sown in sterile sand in
germination boxes as
described in Example I with 10 seeds/treatment/box (total of 60
seeds/treatment). Data was
collected 31 days after sowing. The results are shown in Table 9.
Table 9
Average Seedling Dimensions Produced from Each Treatment
Treatment Normalcy Radical Hypocotyl Cotyledon
Length Length Length
1) Mfd. Seed w/Medium 1 + 40 38.3 % 0.40 cm 1.95 cm 1.01 cm
/L sucrose.
) Mfd. Seed w/Medium 1 + 50 38.3 % 0.63 cm 2.06 cm 1.09 cm
/L sucrose.
Significant differences at a = 0.05 were detected using the PROC GLM program.

CA 02309258 2000-05-08
WO 99/26470 PCTIUS98/24820
-20-
The results show that, for manufactured seeds containing Loblolly pine
embryos,
increasing the sucrose concentration does not increase normalcy of germinants
but can increase
organ elongation of the germinants.
Having illustrated and described the principles of the invention in multiple
embodiments
and examples, it will be apparent to those skilled in the art that the
invention can be modified in
arrangement and detail without departing from such principles. We claim all
modifications coming
within the spirit and scope of the following claims.