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

Definitions

• This invention is directed to methods for the establishment and maintenance of serially propagated cultures of above-ground portions of plants, i.e., stem/leaf or leaf organ cultures.
• the invention is directed to the method for the establishment and maintenance of serially propagated cultures of the above-ground portions of plants.
• a culture of an above-ground plant portion is initiated on a ⁇ nutrient plant cell medium containing a growth regulator.
• the initial culture is maintained under appropriate growth culture conditions after which the culture is transferred to a liquid nutrient plant cell medium containing a growth regulator.
• the culture is maintained in the liquid medium until the cultures without roots are established. These tures grow rapidly and are serially subcultured in liquid medium containing a growth regulator so long as opti ⁇ mum growth continues.
• the method of this invention makes possible the direct propagation of original plant organs without embryogenesis or regeneration of a whole plant with
• a culture of an above-ground plant portion is initiated on a solid plant tissue culture medium con ⁇ taining a growth regulator.
• the selected above-ground plant portion should be comprised of young vigorous dividing tissue from plants after germination.
• the plant portion may be seedlings or excised portions of stem/leaf or leaf organs containing meristematic regions of aseptically or non-aseptically grown plants. Non-aseptic plant portions are sterilized before intro ⁇ duction to the medium.
• a preferred medium is Murashige and Skoog's revised tobacco medium containing agar.
• This solid medium preferably contains a cytokinin- like growth regulator such as kinetin and/or benzyl- adenine, or the like.
• a cytokinin- like growth regulator such as kinetin and/or benzyl- adenine, or the like.
• benzyladenine When benzyladenine is used, it is incorporated into the medium in amount between about 0.05 to 20 pp , and preferably about 2 to 20 ppm. If kinetin is used, the amounts range from about 1 to 100 ppm.
• the initial culture is maintained under aerobic culture growth conditions at a temperature of about 8° to 30° C for a period of about 3 to 5 weeks.
• a liquid medium preferably Murashige and Skoog's medium without agar, containing a cytokinin-like growth regulator ' .
• Benzyladenine may be used in amounts from about 0.05 to 20 ppm, and preferably from about 0.5 to 10 ppm.
• Kinetin may be used in amounts from about .5 to 200 ppm, or preferably about 5 to 100 ppm.
• the liquid cultures are shaken gently to introduce air to the medium.
• the cultures are maintained under appropriate cul ⁇ ture growth conditions at a temperature between about 8° to 30° C and under continuous or intermittent light conditions between about 100 to 10,000 foot candle power.
• the cultures are maintained under a daylight cycle of about 8 to 20 hours duration and ideally about 16 hours duration.
• leaf cultures without roots appear in the medium and grow very rapidly.
• the leaf cultures are serially sub- cultured into liquid medium about every three to twenty weeks. Each subculture will grow very rapidly and is maintained until optimum organ growth ceases.
• the culture transfers are made under aseptic con ⁇ ditions. Once the above-ground plant cultures are initiated, the new cultures are serially propagated from preexisting cultures without involving regenera ⁇ tion of plant callus or intervention of a whole plant. The generation and propagation of only leaf organ can be achieved in relatively short time and relative- ly minimum effort as compared to field growth of the plant. I_n vitro aseptic serial propagation of above- ground plant portions can be carried out without con ⁇ cern for geographic location or time of year.
• the method of this invention may be applied to most existing plant species. It is especially adapted to the subculturing of plant species from which useful materials may be extracted, including Digitalis, Dioscorea, Chrysanthemum, Pyrethrum, Catharanthus, Pinus, Papaver, Yucca, Guayule and other latex bearing plants, and the like.
• Example 1 Digitalis lanata leaf cultures were established from aseptically germinated seeds. Cotyledon seedlings, i.e., those with cotyledons but without true leaves, were
• Example 2 The establishment of shoot cultures of Dioscorea compositae and their ability to synthesize diosgenin were determined. Young stem segments measuring 3-4 mm and with nodes were collected from greenhouse grown D. compositae plants. The explants were washed with a 1:75.0 dilution of benzalkonium chloride for 3 minutes, washed and surface sterilized with 2 per cent sodium hypochlorite for 3 minutes.
• the explants were asepti- cally cut at the edges and cultured on Murashige and Skoog's revised tobacco medium containing 1 per cent agar and supplemented with different concentrations (0.1 ppm, 0.5 ppm, 0.75 ppm, and 1.0 ppm of benzyl ⁇ adenine) .
• the cultures were incubated in vials con ⁇ taining 18 ml of the medium at 24 +_ 2° C and 16 hours of light (750 f.c.) daily. After 2 subcultures of 30 days each, the shoots were transferred to Murashige and Skoog's liquid media with varying concentrations of benzyladenine.
• the suspension cultures were shaken at 75 rpm at 24 +_ 2° C with 18 hour light periods

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• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Cell Biology (AREA)
• Developmental Biology & Embryology (AREA)
• Botany (AREA)
• Environmental Sciences (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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• 1981
  • 1981-05-11 EP EP19810901470 patent/EP0051675A1/de not_active Withdrawn
  • 1981-05-11 WO PCT/US1981/000663 patent/WO1981003255A1/en unknown

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