ASEPTIC SERIAL PROPAGATION OF ABOVE GROUND PORTION OF PLANTS

Abstract: Methods for the establishment and maintenance of serially propagated cultures of above-ground portions of plants, i.e., cultures of stem/leaf or leaf organs containing meristematic regions of plants. A culture of an above-ground plant portion is initiated on a nutrient plant cell medium containing a growth regulator and maintained under appropriate growth culture conditions, after which the culture is transferred to a liquid nutrient plant cell medium containing a growth regulator. The liquid culture is maintained until the establishment of above-ground plant cultures without roots in the medium. These cultures grow rapidly and are serially subcultured in liquid medium containing a growth regulator so long as optimum growth continues. The method may be applied to most existing plant species without regard to geographic location, time of year, or the like.

Description: ASEPTIC SERIAL PROPAGATION OF ABOVE GROUND PORTION OF PLANTS BACKGROUND OF THE INVENTION: FIELD OF THE INVENTION: 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. Plant callus cultures, cell suspension cultures and plant root organ cultures have been successfully established and serially propagated. According to prior techniques, cultures were serially propagated as plant callus cultures or as embryos to regenerate whole plants with roots. However, heretofore, seri¬ ally propagated cultures of above-ground plant por¬ tions have not been successfully established. SUMMARY OF THE INVENTION: Broadly stated, 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 • _ roots. The process is potentially useful for the production of useful substances such as medicinals, chemicals, biochemicals, and the like. The method pro- vides a useful tool for biosynthesis studies. It may be applied to most existing plant species without re¬ gard to geographic location, time of year, or the like. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT: . 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. Although others may be used, 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. 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. Then the cultures are transferred into 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. Preferably, the cultures are maintained under a daylight cycle of about 8 to 20 hours duration and ideally about 16 hours duration. After about 1-1/2 to 2-1/2 weeks, 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. The invention is further illustrated by the follow¬ ing examples: Example 1 Digitalis lanata leaf cultures were established from aseptically germinated seeds. Cotyledon seedlings, i.e., those with cotyledons but without true leaves, were O vΛ*. WI transferred into Murashige and Skoog's revised tobacco medium containing agar and 10 ppm benzyladenine. After about 4 weeks, the cultures were transferred into a 250 ml Erlenmeyer flask containing 50 ml of liquid Murashige and Skoog's medium with 5 ppm benzyladenine and placed on a gyratory shaker (78 rpm) at 25° C. The cultures were grown under a 16 hour daylight cycle (500 f.c. fluorescent Plant-Grow bulbs). In approximately 2 weeks, leaf cultures without roots were established and grew very rapidly, requiring subculturing every 4 weeks. The 4 week growth index (final tissue fresh wt/inoculum fresh wt.) for the leaf cultures was 20 +_ 2.2. The ability of the cultures to produce digoxin upon repeated subculture was established. Digoxin production was significant (9.0 +_ 1.6 mg. per cent dry wt.) in 4 week old cultures and maintained over 10 passages. As high as 44 +_ 3.8 mg. per cent dry weight of digoxin content was present in 12 week old leaf organ cultures. 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 Induction of shoot buds at the nodes was observed in solid media containing all four levels of benzyl- adenine but greater in media with 0.5 and 0.75 ppm supplements. The shoot buds grew to 5 to 8 mm in 30 days and upon subculturing on their supplement adjusted agar medium an additional 1 to 3 cm in 30 days. The shoots upon transfer to their respective liquid media developed distinct differences in their morphological features. Shoot buds transferred from the 0.5 ppm benzyladenine supplemented medium to a medium without benzyladenine grew only during the first 30 day period. In the 0.1 ppm medium, the stem elongation was rapid and similar to the plant vine. In the 0.5 ppm supple¬ mented medium, both stem and leaves thickened and OMF multiple shoot buds developed at the nodes. The tissue grew rapidly. The internodal stem segments did not form callus or shoot buds in the 0.5 ppm medium, but a thickening of the stem portions was observed. Explants cultured on the 0.075 ppm supple¬ mented medium showed less rapid growth with thicken¬ ing of the stem and leaf elongation. Multiple shoot buds also developed in this medium but to a lesser extent than in the 0.5 ppm supplemented medium. In the 1.0 ppm supplemented medium, the stem thickening was maximum and the leaves were thin, elongated and deformed. This medium did not favor multiple shoot bud formation. Shoots with less leaves and elongated stems synthesized low amounts of diosgenin. Shoots with broad leaves and multiple shoot buds synthesized the highest levels of diosgenin. The diosgenin con¬ tent of these cultures was 72% of the in. vivo shoots. It is apparent that many modifications and varia¬ tions of this invention as hereinbefore set forth may be made without departing from the spirit and scope thereof. The specific embodiments described are given by way of example only and the invention is limited only by the terms of the appended claims.

Claims: The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 1. A method for the establishment and mainten¬ ance of serially propagated cultures of above-ground portions of plants, which method comprises: A) initiating a culture of an above-ground plant portion on a nutrient plant cell medium containing a growth regulator, B) maintaining under culture growth condi¬ tions, C) transferring the resulting culture to a liquid nutrient plant cell medium con¬ taining a growth regulator, D) maintaining until establishment of above- ground plant cultures without roots, and E) serially subculturing the resulting rapidly growing cultures in liquid medium as in subparagraph C) . 2. A method according to Claim 1 wherein the above- ground plant portion is comprised of young dividing tissue selected after germination. 3. A method according to Claim 2 wherein the above- ground plant portion is selected from the group consistin Ol.'.? of seedlings and excised portions of stem/leaf, leaves and above-ground meristematic region of plants. 4. A method according to Claim 1 wherein the initial culture is established and maintained on a solid nutrient medium. 5. A method according to Claim 4 wherein said initial culture is maintained on an agar-containing nutrient medium for about 3 to S weeks before trans¬ fer to a liquid medium. 6. A method according to Claim 1 wherein said growth regulator is a cytokinin-like regulator. 7. A method • according to Claim 6- wherein said growth regulator is selected from the group consisting of benzyladenine and kinetin. 8. A method according to Claim 7 wherein said growth regulator is benzyladenine used in the m.edium in amounts between about 0.05 to 20 ppm. 9. A method according to Claim 1 wherein the cultures are grown under light of about 100 to 10,000 foot candles under a daylight cycle of about 8 to 20 hours. OlicI 10. A method according to Claim 1 wherein the liquid medium is maintained under conditions to provide air to the cultures. 11. A method according to Claim 1 wherein the leaf cultures are subcultured about every 3 to 20 weeks, 12. A method according to Claim 1 wherein said plant is selected from a species of the group consist¬ ing of Digitalis, Dioscorea, Chrysanthemum, Pyrethrum, Catharanthus, Pinus, Papaver, Yucca, Guayule and other latex bearing plants. 13. A serially propagated rootless plant produced by the method of Claim 1. OMPI

Inventors: LUI J, STABA E
Applicants: UNIV MINNESOTA
Application year: 1981
Main classification: A01G1
IPC codes: A01H4