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
• • 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
  • A01H4/002—Culture media for tissue culture
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
  • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
  • A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
  • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
  • A01G24/30—Growth substrates; Culture media; Apparatus or methods therefor based on or containing synthetic organic compounds
• • 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
• • 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
  • A01H4/008—Methods for regeneration to complete plants
• • 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
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
  • A01H5/04—Stems
• • 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
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy

Definitions

• the common ash has the widest range. It is native throughout northern Europe, up to 64°N (in Norway), south to the Mediterranean Sea, and east to Russia (almost to the Volga River). The distribution of the other two species is, however, limited to the southern areas (FRAXIGEN. 2005). Due to such a huge range of F. excelsior and often the key importance of ash wood in the economy, the decline of this species constitutes a huge problem, not only ecological, but also an economic one.
• the multi stage approach required in the somatic embryogenesis method results in an increased risk of failure to obtain a sapling, i.a. because each step increases the probability of the culture being infected.
• the multi stage nature also results in greater time-consumption, and, in turn, the greater time-consumption increases the risk of mutation and necessitates the need for costly maintaining of strictly defined conditions and the control thereof for a long time and under periodically changing conditions depending on the cultivation stage. All this means that in the process of obtaining saplings by the method of somatic embryogenesis, spontaneous changes occur in callus cells, which make plants regenerated in this method not as homogeneous as would be expected from a vegetative reproduction method.
• the aim of the invention is to provide a method for the production of saplings of the common ash ( Fraxinus excelsior L), which would allow to avoid the disadvantages of known methods of the production of saplings.
• the subject of the invention is a method for obtaining saplings of the common ash ( Fraxinus excelsior L.) and the composition of the media which can be used in this method, which are defined in detail in the appended claims.
• the essence of the present invention is to enable the use of indirect adventitious organogenesis which omits the stage of shaping and development of the somatic embryo, and thus significantly reduces the number of subcultures and expensive components of culture media.
• the regeneration of the plant takes place directly from the callus tissue, which results not only in lower costs of the production of saplings, but also, very importantly, in their greater genetic stability than in the case of micro-propagation by means of the somatic embryogenesis method.
• Example 1 Obtaining saplings of the common ash ( Fraxinus excelsior L.).
• compositions of the media used are summarized in Table 1 .
• Disinfection takes place in two stages and the indicated amounts are calculated for approx. 250 seeds.
• the wind dispersal apparatus i.e. samara, was removed from good quality seeds (without visible damage) of the common ash.
• Seeds without samaras were placed in a metal, tightly closed strainer with a minimum recommended diameter of 65 mm. In case of a smaller number of seeds, e.g. 100, the recommended minimum strainer diameter is 40 mm.
• Seeds closed in the strainer were placed in a glass beaker filled with a 70% solution of undenatured ethanol in an amount that allowed the seeds to be completely immersed in this solution, and shaken for 30 seconds.
• the seeds in the strainers were transferred to a tall, e.g. 2-liter beaker and washed in a stream of running tap water at a temperature of about 18°C.
• the seeds were taken out of the strainer with the use of sterile tweezers and placed in a sterile glass beaker with a volume of about 400 ml_, and next 250 mL 5% of the previously prepared aqueous polyvinylpyrrolidone (PVP) solution was poured over them.
• PVP polyvinylpyrrolidone
• the beaker with seeds was tightly secured, maintaining sterile conditions, e.g. with sterile aluminum foil and sealed, e.g. by wrapping with parafilm.
• the seeds were disinfected in 8% sodium hypochlorite (NaOCI) solution from Fluka or Sigma- Aldrich (catalog number: 13440), which contains 6-14% active chlorine.
• NaOCI sodium hypochlorite
• the embryos turned “whitish”, lost turgor pressure after sterilization and died.
• the seeds placed in the strainers were shaken vigorously for at least 15 minutes in a NaOCI solution.
• the seeds placed in the strainers were rinsed 5 times in sterile deionized water, each time for at least 1 minute, with changing the water after each rinse.
• the seeds disinfected according to the above procedure were ready for the isolation of zygotic embryos.
• the embryos should be isolated in a laminar chamber immediately after the seeds have been disinfected.
• Zygotic embryos (primary explants) isolated in this way were placed in groups of 10 on a sterile medium 1 (Tab. 1A) in sterile ventilated Petri dishes, in a horizontal position, so that 1 ⁇ 2 of their surface was in contact with the medium.
• a sterile medium 1 Tab. 1A
• the humidity was 40- 45%
• temperature was 23°C +/- 1°C
• the fragments of primary seedlings turned into physiologically developed secondary seedlings (Fig. 3) (with a shoot length of approx. 5-7 mm), well coloured with chlorophyll, with cotyledons and embryonic root (approx. 5 mm long) with an average of 8 secondary seedlings grown in a single Petri dish, in which all the fragments obtained from the five cut primary seedlings with the remainder of the callus tissue had been previously placed.
• the resulting secondary seedlings were sterilely transferred to sterilized crystallizers, preferably with a diameter of 80 mm (preferably 4 seedlings per vessel), closed with a glass lid (the lid may be e.g. the upper part of a Petri dish) or analogous sterile vessels dedicated to plant breeding, e.g. from SPL Life Sciences Co 100 (dia.) x 40 (height) mm, cat. no. 310100, filled with sterile medium 3, less rich than the previous media in selected ingredients (mainly microelements) and enriched with a growth regulator, i.e. IBA auxin (indolylbutyric acid) at a concentration of 5.28 mg/L (for full composition see Tab. 1C).
• a growth regulator i.e. IBA auxin (indolylbutyric acid) at a concentration of 5.28 mg/L (for full composition see Tab. 1C).
• the disclosed invention provides a method for obtaining saplings of the common ash (, Fraxinus excelsior L.) from mature seeds by indirect adventitious organogenesis in a micro-propagation process, taking into account the following composition of the media used at each stage.
• Table 1 The composition of the media for the initiation and proliferation of callus tissue and the adventitious organogenesis of seedlings and saplings of F. excelsior under in vitro conditions.
• the acceptable range of concentrations is given in brackets, with the indication of the preferable value outside the brackets marked ’*’
• Ash dieback one of the worst tree disease epidemics could kill 95% of UK’s ash trees.

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• Life Sciences & Earth Sciences (AREA)
• Developmental Biology & Embryology (AREA)
• Environmental Sciences (AREA)
• Engineering & Computer Science (AREA)
• Botany (AREA)
• Biotechnology (AREA)
• Cell Biology (AREA)
• Physiology (AREA)
• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Inorganic Chemistry (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Fertilizers (AREA)
• Silicates, Zeolites, And Molecular Sieves (AREA)

Applications Claiming Priority (2)

Publications (1)

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Family Applications (1)

Country Status (3)

Families Citing this family (2)

• 2020
  • 2020-03-18 PL PL433288A patent/PL242938B1/pl unknown
• 2021
  • 2021-03-18 WO PCT/PL2021/050017 patent/WO2021187995A2/en not_active Ceased
  • 2021-03-18 EP EP21726222.9A patent/EP4120824A2/en active Pending

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