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 No. 08/219879 (unpublished).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 xe2x80x9cinitiation mediaxe2x80x9d 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 xe2x80x9cmaturation mediumxe2x80x9d 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 xe2x80x9cwoody speciesxe2x80x9d 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 xe2x80x9conxe2x80x9d and xe2x80x9cinxe2x80x9d 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, 500xe2x80x94(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
Preferably said ion concentrations are
In another embodiment preferably also present in the medium are the following inorganic ions or the total presence of inorganic ions is as follows
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
Preferably arginine is about 700, preferably asparagine is about 2,100 and preferably glutamine is about 7,300.
In another aspect the present invention consists in 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 culture) on and/or in an initiation medium, and
allowing sufficient time for the initiation to take place,
wherein the initiation medium contains ABA and amino acids, or
wherein the initiation medium contains amino acids and no ABA.
Preferably conifers are the source of the explants.
Preferably the sufficient time is of the order of about 4-6 weeks.
Preferably the environment is in a sterile tissue culture vessel in a controlled environment at 15-28xc2x0 C.
In a further aspect the present invention consists in a method of initiation of embryogenic tissue from a source of immature conifer or other woody species embryos, 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;
Preferably the sucrose is about 30,000, the gelling agent is GELRITE(copyright) about 4,500, and the abscisic acid is about 15.
In this respect reader is referred to South African Patent Specification No. SA 93/4807 where media is used for maturation only. The present invention recognises the usefulness and advantages of a media (not necessarily including ABA) for initiation and also (when preferably including ABA) for maturation.
In a further aspect the invention is 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 culture 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 initiation 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) at least the initiating medium contains at least one amino acid and no ABA.
Preferably said at least one amino acid is selected from the group Arginine, Asparagine, Glutamine, Citrulline, Ornithine, Lysine, Alanine and Proline.
Preferably the maturation medium contains ABA and at least one amino acid.
The invention also consists in embryos thus matured.
In still a further aspect the present invention consists in 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 ranges in concentration of the more significant ions in a preferred medium.
Preferably said ion concentrations are
Preferably said ion concentrations are as follows
Preferably 5 g/l-50 g/l )w/v) Sucrose is also present (preferably about 30g/l).
Preferably 3-8 grams gellan gum per litre (preferably about 4.5 grams) or other gelling agent is also present.
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
Preferably arginine is about 700, preferably asparagine is about 2,100 and preferably glutamine is about 7,300.
In yet a further aspect the present invention consists in and/or an initiation and maturation media for embryogenic tissue, said medium comprising;
Preferably the sucrose is about 30,000, the gelling agent is GELRITE(copyright) about 4,500, and the abscisic acid is about 15.
In yet a further aspect the present invention consists in an initiation embryogenic medium that is also effective as a maturation medium for initiated and maintained embryogenic tissue resulting from the initiation.
In still a further aspect the present invention consists in a method of increasing the efficiency of maturation of somatic embryos which comprises initiating and maturing the embryogenic tissue on a (preferably substantially common) medium which either
(a) contains ABA and amino acids,
(c) is of a composition substantially as follows;
Preferably the sucrose is about 30,000, the gelling agent is GELRITE(copyright) and is about 4,500, and the abscisic acid is about 15.
Preferably the efficiency is a potentiated increase in the efficiency in that the use of the initiation medium as set out in (c) or containing ABA and amino acids or just amino acids increases the percentage of cell lines produced at initiation of the embryogenic tissue. (FIGS. 1-5). The cell lines initiated by the invention that go on to produce mature somatic embryos is also increased.
The present invention encompasses in the preparation of somatic embryos (particularly of conifers) a merged yet still sequential initiation and maturation procedure using a common or substantially common medium.
The present invention also envisages a procedure of generating mature somatic embryos which comprises:
initiation of embryogenic tissue from a source of immature conifer or other woody species embryos by a procedure of the present invention previously defined, and
maturing at least some of the embryogenic tissue to mature somatic embryos on the same medium but removing some of the initiated embryogenic tissue from the medium for bulking up and/or maintenance on a different media or for long term storage by cryo preservation before proceeding with additional maturation for producing much larger numbers of mature somatic embryos on the same or similar medium.
In a further aspect the present invention consists in mature embryos yielded by a method in accordance with the present invention and/or the use of an initiation and/or maturing media as previously set forth.
In yet a further aspect the present invention consists in a harvested product of a conifer or other woody species where the seed and/or seedling has resulted from the use of a method of the present invention and/or an initiation and/or maturing media as previously set forth.
In still a further aspect the present invention consists in wood, wood chips or cellulosic fibre derived from such a harvested product.
Indeed in prior art procedures the stages are as set out below.
1. Initiation of embryogenic tissue
2. Maintenance of embryogenic tissue (optional cryopreservation)
3. Development of embryogenic tissue
4. Maturation of somatic embryos
5. Starvation and storage of somatic embryos (NZ Patent Appi. No. 272210)
6. Germination of somatic embryos
7. Growth of somatic seedling in greenhouse
8. Growth of somatic seedlings in field
With the adoption of the present invention, initiation and maturation using the same medium, steps 1 to 4 of such a prior art procedure merge into a combined initiation and maturation stage, the duration of which is approximately 8-12 weeks.
1. Initiation and growth of embryogenic tissue (with optional cryopreservation) 1a.
2. Maturation of somatic embryos
3. Starvation and storage of somatic embryos (preferably as in New Zealand Patent Specification No. 272210)
4. Germination of somatic embryos
5. Growth of somatic seedling in greenhouse
6. Growth of somatic seedlings in field
Somatic embryos produced by this invention can be used in the performance of the invention of our New Zealand Patent Specification No. 272210.
The methods and media of the present invention will now be described with particular reference to Pinus radiata as New Zealand""s predominant exotic conifer species.
For initiation, whole megagametophyte are not used because of the uncertainty of whether a seed is fertilised or not. If not fertilised, no useful embryogenic tissue is formed.
Dissected fertilised embryos at the bullet stage and before the pre-cotyledonary stage, about 500-1000 called embryo head developmental stage. Other stages of development may be appropriate for other woody species, depending on the size and morphology of fertilised embryos.
Dissected embryos can be placed directly onto the initiation medium or onto a nurse culture. Vigorous growth of embryogenic tissue results.
Initiation media in at least some preferred forms for Pinus radiata did not contain traditional/conventional plant growth regulators such as auxins and cytokinins (die; 2, 4-D, IAA, NAA, IBA and BAP, 2-IP, Zeatin, TDZ etc). The media of the present invention does contain ABA and/or some or all amino acids.
Typically initiation obtained with this method and of those initiated typically mature. These results have been shown over a range of eight genetically different families of Pinus radiata. 
The performance of the present invention will now be described with particular reference to the accompanying drawings against procedures also for Pinus radiata as disclosed in South African Patent No. 93/4807.
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