Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
1998-01-30
2002-07-09
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C435S430000, C435S430100, C435S431000
Reexamination Certificate
active
06417001
ABSTRACT:
The present invention relates to an improved conifer or other woody species embryogenesis process for initiation and maturation.
The initiation and maturation of embryogenic tissue and somatic embryos respectively has hitherto been part of a stagewise process from which explant to embryogenic tissue through to germination of somatic embryos and growth of somatic seedlings in the field has involved the 8 stages referred to hereinafter.
1. Initiation of embryogenic tissue
2. Maintenance of embryogenic tissue
3. Development of embryogenic tissue
4. Maturation of somatic embryos
5. Starvation and storage of somatic embryos (see New Zealand Patent Specification No. 272210)
6. Germination of somatic embryos
7. Growth of somatic seedling in greenhouse
8. Growth of somatic seedlings in field
Protocols for somatic embryogenesis for conifers typically involve several stages from initiation of embryogenic tissue through to somatic embryo maturation and germination. Patents providing background as to the use of embryogenesis to create somatic embryos include WO95/14373, U.S. Pat. No. 5036007, U.S. Pat. No. 5,034,326, U.S. Pat. No. 5,041,382, U.S. Pat. No. 4,957,866, AU 37150/93, U.S. Pat. No. 5,294,549, South Africa 93/4807, and U.S. (CIP) Ser. No. 08/219879 (unpublished).
For initiation of conifer embryogenic cell lines whole gametophytes containing immature fertilised embryos or dissected immature fertilised embryos are used as explants. Explants are placed on several different “initiation media” to initiate embryogenic tissue either with or without growth regulators. The average percentage initiation over an entire seasonal initiation for radiata pine using whole gametophytes as explants has varied from 5-10%. The best percentage initiation has varied from 6-34% for the best developmental window of sampling the explants from each seedlot. An initiated cell line is defined as an established and maintained cell line.
For maturation of conifer somatic embryos, initiated embryogenic cell lines are placed onto several media to maintain and develop the embryogenic tissue and multiply the number of embryo initials. Embryogenic tissue is then generally placed on a “maturation medium” to encourage the tissue to form mature embryos. Typically this maturation media contains abscisic acid (ABA). The percentage of initiated cell lines that are able to continue growth and form mature somatic embryos is typically 1-10%. Higher percentages of up to 25% have been obtained when a selection of 20-25% of the maintained embryogenic cell lines are placed on maturation media, i.e.; not all cell lines initiated are subsequently suitable for placement on maturation media.
The percentage of cell lines that are currently initiated using previously patented or published protocols is too low for adequate clonal representation within a family or cross and genetic diversity is not maintained satisfactorily.
Furthermore, from the embryogenic cell lines initiated the representation of clones forming mature embryos within a family is further reduced.
It is desirable from the perspectives of clonal testing, genetic diversity, process efficiency and cost effectiveness to have at least 50% initiation of clonal embryogenic cell lines and at least 30% formation of mature somatic embryos from those initiated embryogenic cell lines.
The present invention can achieve or at least approach this.
The present invention relates to various procedures and related methods and includes an embryogenic initiation medium which will result in changes to at least stages 1, the prospect of a merging of steps 2 and 3 with each of stages 1 and 4 and additionally potentiates the outcome at maturation step 4 for that embryogenic tissue capable of generating somatic embryos, the present invention therefore providing an increased efficiency over the prior art procedures.
It is to this that the present invention is directed.
In a first aspect the present invention consists in 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 culture itself on or in the initiating medium, and
allowing sufficient time for initiation to take place,
wherein (i) the initiation medium contains ABA and/or at least one amino acid, or (ii) the initiation medium containing just amino acids.
As used herein “woody species” includes the groups of species Eucalyptus family, Proteaceae, Myrtaceae, Rosaeceae, Punicaceae, etc.
Preferably said conifer immature zygotic embryo explants for the invention include those of
Pinus radiata
(or Monterey pine), hybrids of
Pinus radiata
and genetically modified
Pinus radiata
. This procedure is also applicable to other conifer species, viz, loblolly pine, Douglas fir, spruce species etc. and, of course, hybrids or genetically modified versions thereof.
Reference to “on” and “in” in respect of the media at least contemplates the use of gelled and/or liquid media.
Preferably said explant is not the whole megagametophyte and preferably is the dissected fertilised embryos at the bullet stage and before the pre-cotyledonary stage, 500—(if radiata pine) celled embryo head developmental stage and most probably different called embryo head counts for other species which have different sized and shaped embryos.
Preferably the initiation medium does not contain traditional/conventional plant growth regulators such as auxins and cytokinins (eg; 2, 4-D, IAA, NAA, IBA, BAP, 2-IP, Zeatin, TDZ, etc). Nor preferably is it a medium for initiation with no growth regulators as outlined in South Africa 93/4807. But it does contain ABA and/or one or more amino acids.
Preferably ABA (Abscisic Acid) is present.
Preferably where said initiating medium is also to be used as the maturation medium ABA is present.
In another preferred form Abscisic Acid (ABA) may be absent but at least one amino acid is present. Preferablv said amino acid that is present is one or more of the amino acids Arginine, Asparagine. Glutamine, Citrulline, Ornithine, Lysine, Alanine and Proline.
Preferably Glutamine is present.
Preferably at least one of Asparagine and Arginine is also present.
Preferably Glutamine, Asparagine and Arginine are present.
Preferably the initiation medium contains both ABA and at least one of the aforementioned amino acids and optionally several or all of the aforementioned amino acids.
Preferably said initiation medium includes in addition to said ABA and/or said at least one amino acid and other nutrient sources such as, for example, a source of essential macro and micro elements, vitamins, carbohydrates, inositol etc.
Preferably the initiation medium includes inorganic ions in the following ranges in concentration of the more significant ions in a preferred medium
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
Preferably 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
In another embodiment preferably also present in the medium are the following inorganic ions or the total presence of inorganic ions is as follows
ION
CONCENTRATION (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
Preferably also included are 5 g/l-50 g/l (w/v) Sucrose (preferably about 30 g/l).
Preferably it also includes 3-8 grams gellan gum per litre (preferably about 5 grams) or other gelling agent (eg; agar or other).
Preferably it also includes 5 mg/l-50 mg/l (w/v) Abscisic acid (ABA) (preferably about 15 mg/l).
Preferably the amino acids are present in the following ranges
ION
CONCENTRATION RANGE
Aitken-Christie Jennifer
Parkes Bryan Donald
Carter Holt Harvey Limited
Jacobson & Holman PLLC
Lankford , Jr. Leon B.
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