US2003022372A1PendingUtilityA1
Embryogenesis process for initiation and maturation
Est. expiryMay 25, 2015(expired)· nominal 20-yr term from priority
A01H 4/00
28
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Claims
Abstract
The present invention is a method of producing mature somatic conifer embryos at a higher yield. Explants of immature embryos are placed on or in an initiation medium or on a nurse culture which is on or in the initiation medium. Over a period of time, initiation takes place and the initiated embryos are ultimately matured on an appropriate maturation medium. The initiation medium and the maturation medium may be the same or different but the initiation medium must contain at least ABA and at least one amino acid or at least one amino acid and no ABA. The maturation medium must contain ABA and at least one amino acid. Advantages of the present invention include ensuring greater efficiency in both initiation and maturation of any initiated embryogenic tissue.
Claims
exact text as granted — not AI-modified1 . A method of initiating embryogenic tissue from a source of immature embryos of a conifer or other woody species, said method comprising:
placing explants of the immature embryos on and/or in an initiation medium or on a nurse callus itself on or in the initiating medium, and allowing sufficient time for initiation to take place, wherein the initiation medium contains
(i) at least one amino acid, or
(ii) both ABA and at least one amino acid.
2 . A method of claim 1 wherein the source is a conifer.
3 . A method of claim 1 or 2 wherein said explant is not the whole megagametophyte and is the dissected fertilised embryos at 2 to 500 or more—(if a conifer species) celled embryo head stage.
4 . A method of claim 3 when dissected at about the bullet or 500-1000-celled embryo head stage.
5 . A method of any one of the preceding claims wherein the initiating medium does not contain traditional/conventional plant growth regulators such as auxins and cytokinins (eg; 2, 4D, IAA, NAA, IBA, BAP, 2-IP, Zeatin, TDZ, etc) but it does contain ABA and/or one or more amino acids.
6 . A method of any one of the preceding claims wherein ABA (Abscisic Acid) is present.
7 . A method of any one of claims 1 to 5 wherein ABA is absent but at least one amino acid is present being one or more of said amino acid(s) Arginine, Asparagine, Glutamine, Citrulline, Ornithine, Lysine, Alanine and Proline.
8 . A method of claim 6 or 7 wherein Glutamine is present.
9 . A method of claim 6 , 7 or 8 wherein at least one of Asparagine and Arginine is present.
10 . A method of claim 6 or 7 wherein Glutamine, Asparagine and Arginine are present.
11 . A method of any one of the preceding claims wherein said initiating medium includes in addition to said ABA and/or said at least one amino acid other nutrient sources such as, for example, a source of essential macro and micro elements, vitamins, carbohydrates, inositol etc.
12 . A method of any one of the preceding claims wherein the initiation medium includes inorganic ions in the following concentrations
CONCENTRATION RANGE
ION
(mmoles/l)
NO 3
4-27
NH 4
0.5-6.8
Ca
0-0.9
Fe
0-0.15
Na
0-7
Zn
0-0.135
Cu
0-0.05
Mg
0-3.24
13 . A method of claim 12 wherein said ion concentrations are
CONCENTRATION RANGE
ION
(mmoles/l)
NO 3
about 17.8
NH 4
about 1.96
Ca
about 0.17
Fe
about 0.10
Na
about 3.85
Zn
about 0.09
Cu
about 9.61 × 10 −3
Mg
about 1.62
14 . A method of any one of claims 1 to 11 wherein also present in the medium are the following inorganic ions or the total presence of inorganic ions is as follows
CONCENTRATION
ION
(mmoles/l)
NO 3
17.80
NH 4
1.96
TOTAL
19.76
N
P
1.96
K
14.16
Ca
0.17
Mg
1.62
Cl
3.42 × 10 −1
Fe
0.10
S
1.83
Na
3.85
B
0.13
Mn
1.62 × 10 −2
Zn
0.09
Cu
9.61 × 10 −3
Mo
8.27 × 10 −4
Co
8.41 × 10 −4
I
6.02 × 10 −3
15 . A method of any one of the preceding claims wherein the media contains 5 g/l-50 g/l (w/v) Sucrose.
16 . A method of any one of the preceding claims wherein the media contains 3-9 grams gellan gum or other gelling agent.
17 . A method of any one of the preceding claims wherein the amino acids are present in the following ranges
CONCENTRATION RANGE
ION
(mg/l)
Arginine
500-2,000
Asparagine
1,000-4,000
Glutamine
1,000-10,000
Citrulline
0-50
Ornithine
0-50
Lysine
0-50
Alanine
0-50
Proline
0-50
18 . A method of any one of the preceding claims including allowing at least partial maturation wherein said initiation media is used as the maturation media.
19 . A method of initiation of embryogenic tissue from a source of immature conifer or other woody species, said method comprising:
placing explants of the immature fertilised embryos (directly or indirectly eg; nurse callus) on and/or in an initiation medium, and allowing sufficient time for the initiation to take place, wherein the initiation medium contains
(i) amino acids, or
(ii) ABA and amino acids.
20 . A method of claim 19 wherein conifers are the source of the explants.
21 . A method of claim 19 or 20 wherein the initiation medium contains ABA.
22 . A method of any one of claims 19 to 21 wherein the sufficient time is of the order of about 4 weeks.
23 . A method of any one of claims 19 to 22 wherein the environment is in a sterile tissue culture vessel at 15-28° C.
24 . A method of initiation of embryogenic tissue from a source of immature conifer or other woody species, said method comprising:
placing explants of the immature fertilised embryos (directly or indirectly eg; nurse cells) on and or in an initiation medium, and allowing sufficient time for the initiation to take place, wherein the initiation medium is; Final Rate per Litre (mg) Potassium Nitrate (KNO 3 ) 1431 Magnesium Sulphate (MgSO 4 .7H 2 O) 400 Sodium Nitrate (NaNO 3 ) 310 Ammonium Dihydrogen (NH 4 H 2 PO 4 ) 225 Phosphate Calcium Chloride (CaCl 2 .2H 2 O) 25 Zinc Sulphate (ZnSO 4 .7H 2 O) 25 Boric Acid (H 3 BO 3 ) 8.0 Manganese Sulphate (MnSO 4 .H 2 O) 2.72 Copper Sulphate (CuSO 4 .5H 2 O) 2.4 Potassium Iodide (KI) 1.0 Cobalt Chloride (CoCl 2 .6H 2 O) 0.2 Molybdic Acid (Na 2 MoO 4 .2H 2 O) 0.2 EDTA-Disodium Salt 40 Iron Sulphate 7H 2 O 30 Nicotinic Acid 5.0 Thiamine HCl 5.0 Pyridoxine 0.5 Inositol 1000 Arginine 700 Asparagine 2100 Glutamine 7300 Citrulline 3.95 Ornithine 3.80 Lysine 2.75 Alanine 2.0 Proline 1.75 Abscisic Acid 5 to 50 Sucrose 5,000 to 50,000 Gelling Agent (GELRITE ™) 3,000 to 8,000
25 . A method of producing mature somatic embryos comprising the steps
(1) placing explants of the immature embryos on and/or in an initiation medium or on a nurse callus itself on or in the initiation medium, (2) allowing the initiation to take place, (3) (whether after optional storage and maintenance or not) maturing the initiated embryos on an appropriate maturation medium, and wherein
(a) the initiating and maturation medium may be the same or different,
and wherein
(b) (i) at least the initiating medium contains ABA and at least one amino acid, or
(b) (ii) the initiating medium contains no ABA but includes at least one amino acid.
26 . A method of claim 25 wherein the source is a conifer.
27 . A method of claim 26 wherein ABA is present.
28 . A method of claim 25 , 26 or 27 wherein said at least one amino acid is selected from the group Arginine, Asparagine, Glutamine, Citrulline, Ornithine, Lysine, Alanine and Proline.
29 . A method of any one of claims 25 to 28 wherein the maturation medium contains ABA and at least one amino acid.
30 . Embryos matured by a method or using a method of any one of the preceding claims.
31 . An initiation and/or maturation media for embryogenic tissue, said medium comprising in addition to a presence of ABA and at least one amino acid, inorganic ions in the following concentrations
CONCENTRATION RANGE
ION
(mmoles/l)
NO 3
4-27
NH 4
0.5-6.8
Ca
0.01-0.9
Fe
0.025-0.15
Na
0.5-7
Zn
0.023-0.135
Cu
6 × 10 −4 -5 × 10 −2
Mg
0.405-3.24
32 . A media of claim 31 wherein said ion concentrations are
CONCENTRATION RANGE
ION
(mmoles/l)
NO 3
about 17.8
NH 4
about 1.96
Ca
about 0.17
Fe
about 0.10
Na
about 3.85
Zn
about 0.09
Cu
about 9.61 × 10 −3
Mg
about 1.62
33 . A media of claim 31 or 32 wherein said ion concentrations are as follows
CONCENTRATION
ION
(mmoles/l)
NO 3
17.80
NH 4
1.96
TOTAL
19.76
N
P
1.96
K
14.16
Ca
0.17
Mg
1.62
Cl
3.42 × 10 −1
Fe
0.10
S
1.83
Na
3.85
B
0.13
Mn
1.62 × 10 −2
Zn
0.09
Cu
9.61 × 10 −3
Mo
8.27 × 10 −4
Co
8.41 × 10 −4
I
6.02 × 10 −3
34 . A media of any one of claims 31 ro 33 wherein 5 g/l-50 g/l)w/v) Sucrose is also present.
35 . A media of any one of claims 31 to 34 wherein 3-9 grams gellan gum per liter is present.
36 . A media of any one of the preceding claims wherein amino acids are present in the following ranges
ION
CONCENTRATION RANGE (mg/l)
Arginine
500-2,000
Asparagine
1,000-4,000
Glutamine
1,000-10,000
Citrulline
0-50
Ornithine
0-50
Lysine
0-50
Alanine
0-50
Proline
0-50
37 . An initiation and maturation media for embryogenic tissue, said medium comprising;
Final Rate per Litre
(mg)
Potassium Nitrate
(KNO 3 )
1431
Magnesium Sulphate
(MgSO 4 .7H 2 O)
400
Sodium Nitrate
(NaNO 3 )
310
Ammonium Dihydrogen
(NH 4 H 2 PO 4 )
225
Phosphate
Calcium Chloride
(CaCl 2 .2H 2 O)
25
Zinc Sulphate
(ZnSO 4 .7H 2 O)
25
Boric Acid
(H 3 BO 3 )
8.0
Manganese Sulphate
(MnSO 4 .H 2 O)
2.72
Copper Sulphate
(CuSO 4 .5H 2 O)
2.4
Potassium Iodide
(KI)
1.0
Cobalt Chloride
(CoCl 2 .6H 2 O)
0.2
Molybdic Acid
(Na 2 MoO 4 .2H 2 O)
0.2
EDTA-Disodium Salt
40
Iron Sulphate 7H 2 O
30
Nicotinic Acid
5.0
Thiamine HCl
5.0
Pyridoxine
0.5
Inositol
1000
Arginine
700
Asparagine
2100
Glutamine
7300
Citrulline
3.95
Ornithine
3.80
Lysine
2.75
Alanine
2.0
Proline
1.75
Abscisic Acid
5 to 50
Sucrose
5,000 to 50,000
Gelling Agent (GELRITE ™)
3,000 to 8,000
38 . An initiation embryogenic medium that is also effective as a maturation medium for initiated and maintained embryogenic tissue resulting from the initiation, said medium being as claimed in any one of claims 31 to 37 .
39 . A method of increasing the efficiency of maturation of somatic embryos which comprises initiating and maturing the embryogenic tissue on a substantially common medium which either
(a) contains ABA and amino acids, (b) is of a composition substantially as follows; Final Rate per Litre (mg) Potassium Nitrate (KNO 3 ) 1431 Magnesium Sulphate (MgSO 4 .7H 2 O) 400 Sodium Nitrate (NaNO 3 ) 310 Ammomum Dihydrogen Phosphate (NH 4 H 2 PO 4 ) 225 Calcium Chloride (CaCl 2 .2H 2 O) 25 Zinc Sulphate (ZnSO 4 .7H 2 O) 25 Boric Acid (H 3 BO 3 ) 8.0 Manganese Sulphate (MnSO 4 .H 2 O) 2.72 Copper Sulphate (CuSO 4 .5H 2 O) 2.4 Potassium Iodide (KI) 1.0 Cobalt Chloride (CoCl 2 .6H 2 O) 0.2 Molybdic Acid (Na 2 MoO 4 .2H 2 O) 0.2 EDTA - Disodium Salt 40 Iron Sulphate 7H 2 O 30 Nicotinic Acid 5.0 Thiamine HCl 5.0 Pyridoxine 0.5 Inositol 1000 Arginine 700 Asparagine 2100 Glutamine 7300 Citrulline 3.95 Ornithine 3.80 Lysine 2.75 Alanine 2.0 Proline 1.75 Abscisic Acid 5 to 50 Sucrose 5,000 to 50,000 Gelling Agent (GELRITE ™) 3,000 to 8,000
40 . A method of claim 40 wherein (a) or (c) is used.
41 . A method of claim 40 or 41 wherein (c) is used and the sucrose is about 30,000 and the gelling agent is GELRITE™ about 4,500.
42 . A method of any one of claims 39 to 41 wherein the efficiency is a potentiated increase in the percentage of clones producing mature embryos from embryogenic tissue, the embryogenic tissue having been initiated as in claim 1 - 24 .
43 . A method of any one of claims 39 to 42 wherein the efficiency is as a result of reduced contamination owing to the use of the same media for initiation and maturation.Join the waitlist — get patent alerts
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