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Tetraploid plants of Artemisia annua L. exhibiting high-artemisinin-yield were successfully induced by treating excised leaves of in vitro plant with 0.1% colchicine. The chromosome number of original diploid plant was confirmed to be 2n=2x=18 whereas that of the tetraploid plant was 2n=4x=36. Morphological and anatomical characteristics of tetraploid plants were obviously different from the diploid counterpart. The tetraploid plants had larger sizes of root system, stomata and glandular secretory trichomes than diploid plant, whereas leaf size in tetraploid was smaller but thicker than diploid. The highest amount of artemisinin content was produced at flower blooming stage in both diploid (2.4% dry weight) and tetraploid (3.8% dry weight) lines. Based on the maximum yield of artemisinin, the optimum harvest time for the diploid lines was flower initiation stage but it was at full blooming stage for tetraploid lines. The highest artemisinin yield in tetraploid plants up to 3.0 mg plant−1 was detected at this stage which was 1.5 times greater than diploid plants. This is a first report that clearly showed the potential of chromosome doubling strategy to produce high-yield line of A. annua plant for artemisinin production.

Establishment of the efficient method for isolating the female germ unit (FGU; egg, synergid and central cell) is useful for the studies on the characterization of each FGU as well as in vitro fertilization and gametosomatic hybridization. In this study, an easy one-step enzymatic procedure was successfully developed to isolate FGU from ovules of Petunia hybrida without releasing the somatic protoplasts from ovular tissues, which could not be achieved in the previous studies. Each FGU was separately liberated after treating the ovules, which were collected from ovaries of flowers one day after anthesis, with an appropriate enzyme solution comprised of 10 g l−1 Cellulase Onozuka R-10, 10 g l−1 Macerozyme R-10, 0.6 M mannitol, 5 mM 2-morpholinoethane-sulfonic acid and 5 g l−1 potassium dextran sulfate, pH 5.8 with 50-rpm shaking for 2 h at room temperature. Isolated FGUs were distinguished by their specific size and characteristics. Fluorescent staining with 4′, 6-diamidino-2-phenylindole could identify the polar nuclei of central cell and the nuclear polarity of egg apparatus cells. After transfer into washing solution supplemented with 0.6 M mannitol using a micropump-connected microcapillary, about 80% of the isolated FGUs were viable for up to 8 h after the isolation, as determining by fluorescein diacetate staining.

Meristematic nodular calli (NOD) were successfully induced from various plant parts such as seeds, flower organs, shoot apices, stem segments and bulb scales of Lilium spp. on media containing 1mg/l picloram alone or in combination with 1mg/l cytokinin. In L. longiflorum, green friable callus (FC) was also induced from bulb scales. Suspension cultures established from both NOD and FC showed high proliferation rate. NOD retained high regeneration ability as well as stable ploidy level during the culture for more than 4 years, while the suspension culture of FC became tetraploid. Protoplasts were efficiently isolated from NOD in some species and cultivars after long term subculture in suspension cultures and the plants were successfully regenerated from the protoplasts of L. x formolongi.

Adventitious shoots and roots were directly formed from young flower bud explants of Agapanthus africanus on Murashige and Skoog (MS) medium containing 30g·L-1 sucrose, 2g·L-1 gellan gum, and kinetin or thidiazuron at 1mg·L-1 in combination with 1mg·L-1 NAA. Friable calli with a high rate of plant regeneration were also induced by culturing the explants on medium containing NAA, picloram or 2, 4-D at concentrations varying from 1 to 10mg·L-1, especially at low concentrations of picloram or 2, 4-D. Shoots were readily induced from these calli when transferred onto plant growth regulator-free or 1mg·L-1 zeatin-containing MS medium. Nodular compact calli were also formed with friable calli on medium supplemented with 2, 4-D or picloram. The compact calli induced with 1mg·L-1 picloram developed into somatic embryos. In both shoot and embryo regeneration systems, complete plantlets were obtained when they were transferred onto a medium containing no plant growth regulator.