Strandberg J. O., Plant Cell, Tissue and Organ Culture 32: 277-282 (1993)
Summary
Meristems of Ophiopogon japonicus (Liliaceae) were grown on a modified MS medium without auxins or cytokinins and plantlets and embryogenic callus were obtained at a low frequency. When meristems growing on modified basal medium were briefly soaked in 0.54 μM naphtaleneacetic acid (NAA) or temporarily grown on medium that contained NAA or NAA and benzyladenine, a larger proportion of meristems developed into plantlets or produced callus and additional plantlets following their return to basal medium. Calluses grown in liquid culture without auxins or cytokinins produced abundant single cells and cell aggregates. Larger cell aggregates formed embryo-like structures that produced roots, cotyledons, and then plantlets following transfer to solid medium. Prolonged liquid culture produced embryo-like structures directly in liquid medium. These structures met many of the criteria for somatic embryos and developed into normal plantlets when placed on solid medium.
Objectives
Production and in vitro propagation of pathogen-free Ophiopogon japonicus plants.
Materials
Plants were obtained from a commercial nursery.
Explant
Meristem tissue was excised 1-2 mm below the point of attachment of the smallest recognizable leaf primordia.
Initial culture
Meristems were removed from MMS (176 mg 1-1 CaC12, 10mg 1-1 inositol, and 20 g 1-1 sucrose) after 30 days growth, soaked for 2 h in a sterile aqueous solution of 0.54 μM naphthalene-acetic acid (NAA) or growth for 14 days on MMS with NAA or NAA and BA, then placed on fresh MMS medium.
Shoot multiplication
Small pieces (2-3 mm diam) of callus produced from cultured meristems were placed in 40 ml of liquid MMS in 250 ml flasks. These cultures were maintained on an orbital shaker at 100 rotations/min in low light intensity (16-h photoperiod, 14 μmol m-2 s-1) at 22°C. Every 2-3 weeks the cultures were sieved through a sterile screen (0.5 mm openings) to remove the large cell aggregates. To initiate embryo cultures, the large cell aggregates added to flasks that contained fresh MMS. Embryogenic callus and embryo-like structures that developed in liquid cultures were placed on solid MMS in plastic petri plates or vials.
Rooting
Acclimation
Plantlets regenerated were planted in a steamed peatvermiculite medium, (Metro Mix 500®, Grace Sierra Hort. Products Co., Milpitas, CA), held in clear plastic containers at high relative humidity (RH) for 1-3 weeks, then moved to a controlled environment room at 22°C, 16-h photoperiod (Cool-White® fluorescent light, 50 μmol m-2 s-1), and finally, to a 20-24°C greenhouse.
Planting
Cultivation conditions
Regenerated plants growing in a greenhouse
Traints of regenerants
Regenerated plants growing in a greenhouse and closely observed for 24 months, there were no abnormalities or apparent differences from the original source plants.