Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
  • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/04—Plant cells or tissues

Definitions

• This invention concerns the cultivation of conifers and relates to a method of producing embryogenic callus from conifer explants and a method of producing plantlets.
• organogenesis involves taking a suitable explant and cultivating the tissue in culture media containing carefully controlled nutrient and hormone levels, resulting in the formation of callus. This callus can then be placed on budding media. The resulting buds are separated and can be grown to form individual plantlets (as described by von Arnold, 1984).
• somatic embryogenesis can only be performed using embryos or young cotyledon as starting materials.
• success has been achieved with cotyledon explants from seven day old Picea abies seedlings (Lelu et al. , 1984) and 30 day old seedlings of Picea mariana and Picea galuca (Attree et al ., 1990) but not from older trees.
• organogenesis is fully successful only when the explant is taken from very young seedlings. When explants from older plants are used, it is possible to obtain adventitious buds, but these cannot be grown into plantlets (Jansson & Born an, 1983; von Arnold, 1984).
• the present invention aims to overcome this problem.
• the invention provides a method of producing embryogenic conifer callus, comprising culturing somatic tissue capable of growth derived from a conifer plant that has produced a resting bud so as to form non-embryogenic callus and then culturing said non-embroygenic callus in the presence of embryogenic callus in a nurse culture so as to produce embryogenic callus.
• Resting buds are produced at the end of the growing season.
• a conifer plant in accordance with the invention defined above which has produced a resting bud must have reached the end of at least one growing season.
• somatic tissue capable of growth is an explant taken from a needle, bud, shoot or root tissue.
• This tissue may be taken from a mature tree, allowing for the selection of elite specimens for cloning.
• the age of the tree may vary for different species but the technique has been successfully performed using explants from a 26 year old specimen of Picea abies.
• "Mature” is understood in this context to mean sexually mature. However, as is well known to those skilled in the art, a mature tree may not have flowered, because Norway Spruces generally flower together, every 5-8 years. Thus a tree may be capable of flowering (and therefore "mature") but will not do so until the appropriate time. Thus a Norway Spruce may first flower at about 15 years old but this is exceptional. Trees are generally 20-25 years old at first flowering .
• Nurse cultures in general are well known to those skilled in the art, being, for example used in transformation experiments.
• embryogenic callus of juvenile origin and the non-embryogenic callus are located in close proximity on opposed sides of a microporous membrane such that there is no direct contact between the two calli and the only communication is via the pores of the membrane. Growth and maintenance of the calli is supported by a suitable culture medium.
• the pores of the membrane are 0.22 microns in diameter.
• the membrane might be nitrocellulose or other suitable material, as appreciated by those skilled in the art.
• the nurse culture comprises embryogenic tissue of the same species as that from which the non-embryogenic callus is derived, but the embryogenic tissue may be from a different strain or species as will be clear to those skilled in the art.
• the invention thus provides a method of producing plantlets, comprising culturing embryogenic callus produced by the method defined above in a suitable manner to produce plantlets.
• the invention provides a method of producing conifer plantlets comprising culturing somatic tissue capable of growth derived from a conifer plant that has produced a resting bud so as to form non-embryogenic callus; culturing said non-embryogenic callus in the presence of embryogenic callus in a nurse culture so as to produce embryogenic callus; and culturing said produced embryogenic callus in a suitable manner so as to generate plantlets.
• Figure 1 shows a photograph of embryogenic callus produced by the method according to the invention.
• ES contained the following (in u oles per litre), KN0 3 (8400) MgS0 4 7H 2 P0 4 (1200). CaCl 2 .2H 2 0 (520), H 4 N0 3 (15000) .ZnS0 4 .7H 2 0 (5), MnS0 4> 4H 2 0 (5) H3BO3 (5) KI (2.3), Na 2 M00 4 .5H 2 0 (0.05), CuS0 4 .5H 2 0 (0.008), C ⁇ Cl 2 .6H 2 (0.008), Na 2 EDTA.2H 2 O(0.03) FeS0 4 -7H 2 0 (0.03), Mesoinitositol (500) Nicotinic Acid (0.8) Thia ine (1.4) Pyrodoxine HCl (0.5) Glycine (2.6) Glutamine (0.002) Naphylacetic acid (10) Benzylaminopurine (5) plus 88m moles of Sucrose
• Embryonic shoot explants were also used as explants from the mature tree dissected from vegetative buds. These explants were collected during the dormant period (from October to April) and are best described as embryonic shoots comprising a meriste , many needle pri ordia, and the crown. Explants were transferred regularly each six weeks to the same medium as was any resultant callus. After six or more transfers from establishment, any non- emoryogenic calli produced was put into a nurse culture with embryogenic callus.
• Nurse cultures comprised embryogenic callus from a line produced by Joachim Buttgereit in 1989 from zygotic embryo explants from seed of a Gerlitz provenance (which can be routinely induced to produce somatic embryos) and a needle or bud non- embryogenic callus physically separated from the embryogenic callus by a 0.22u millipore filter membrane as shown in Figure 1. The same media were used throughout. Nurse cultures were transferred to fresh media each four weeks with great care not to mix the two calli. All cultures were maintained at 22°C in darkness.
• Table 1 summarises the number of expiants which produced non-embryogenic callus after three transfers. All callus arose from the stem heel end of needles and it is clear from Table 1 that the age of the plant from which explants were taken influenced the ability to produce callus. The bud explants taken directly from the 26 year old tree were more productive than needle explants from rooted cuttings. During subsequent transfers the number of calli declined until, at transfer six, 98 of the 139 axenic shoot cultures which originally produced non-embryogenic callus remained. 2 of the 6 year old trees had no explants with non-embryogenic callus and, of the 180 calli from the bud explants taken from the 26 year old tree, 51 survived.
• Nurse cultures were then established from a representative sample of the non-embryogenic calli. After 8 to 10 transfers, embryogenic musilaginous sectors appeared in eight cultures representative of 3 of the axenic shoot culture clones and all those of the 6 year old trees which had surviving calli. Shown in Figure 1, embryogenic callus 10 is easily distinguished as fast growing and spreading in growth habit. Its identity was confirmed by the presence of proembryos and suspensor-like cells after staining and light microscopy. All the new embryogenic callus has been isolated away from the original non- embryogenic material. Nurse cultures allowed non- embryogenic callus to survive: only six calli representing two of the axenic shoot culture clones still survived after twelve passages (without having been put into nurse cultures).
• Somatic embryogenesis is favoured by the use of explants from the youngest tissue available.
• results presented here show that at low frequency embryogenic callus can be produced from explants from older trees.
• the nurse cultures employed promoted growth and survival of the slow growing callus obtained from older trees and promoted the formation of the embryogenic callus.
• the most productive group of explants was from the 6 year old trees, with 5 of the 96 nurse cultures established resulting in embryogenic callus production.

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• Life Sciences & Earth Sciences (AREA)
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• Biotechnology (AREA)
• Health & Medical Sciences (AREA)
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• Wood Science & Technology (AREA)
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• Developmental Biology & Embryology (AREA)
• Organic Chemistry (AREA)
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• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
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• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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• 1992
  • 1992-06-04 GB GB9211991A patent/GB2267714B/en not_active Expired - Fee Related
  • 1992-06-09 CA CA002070846A patent/CA2070846A1/en not_active Abandoned
• 1993
  • 1993-06-01 AT AT0903693A patent/AT403233B/de not_active IP Right Cessation
  • 1993-06-01 DE DE4392367A patent/DE4392367C2/de not_active Expired - Fee Related
  • 1993-06-01 DE DE4392367T patent/DE4392367T1/de active Pending
  • 1993-06-01 WO PCT/EP1993/001365 patent/WO1993023990A1/en active Application Filing
• 1994
  • 1994-11-30 SE SE9404165A patent/SE503845C2/sv not_active IP Right Cessation
  • 1994-12-02 FI FI945682A patent/FI945682A/sv not_active Application Discontinuation

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