direct sunlight in a 22 circC (71.6 °F) greenhouse and watered only after the medium dried. No shoots formed on root pieces that were less than 5 mm in diameter, however 56% of root pieces greater than 5 mm in diameter produced one or more shoots. On average, responding roots produced 2.5 buds and 1.1 elongating shoots. Buds were visible on root pieces 12 weeks after planting and shoot elongation was evident after 16 weeks (Fig. 2B).

MICROCUTTINGS. Preliminary experiments to root microcuttings of pawpaw have met with little success (S.T. Kester and R.L. Geneve, unpublished). In these experiments, microshoots were developed from pawpaw cultures maintained for over 2 years. The original explants were shoots developed on root pieces from the A10-11 understock. Initial treatments placing explants on one-half strength MS (Murashige and Skoog, 1962) salts medium containing IBA (0.49 to 4.9 μM) resulted in a small percentage (3.0%) of microcuttings developing one to two roots per cutting at the 4.9 μM IBA level (Fig. 2C). However, these rooted shoots did not thrive during the acclimatization stage and failed to develop further. Several other protocols used for other difficult-to-root species were tried on a limited basis for pawpaw. Similar to what was done with redbud, pawpaw microshoots were exposed to high levels of IBA (100 μM) for 1 or 2 weeks followed by transfer to in vitro (without auxin) or ex vitro conditions (Yusnita et al., 1990). Similar to what was done with atemoya cultures, cuttings of etiolated pawpaw microshoots in culture were subjected to either solid or liquid MS medium moved to a liquid medium containing 245 μM IBA for 3 d before moving to MS medium with 0.25% activated charcoal (Rasai et al., 1994). These treatments were not more effective than the original in vitro rooting experiments with pawpaw.

tive than the original in vitro rooting experiments with pawpaw.

Initial experiments using seedling explants of pawpaw demonstrated that nodal explants responded more favorably than apical sections for establishment of cultures (Finneseth, 1997). When nodal explants were treated with a range of benzyladenine (BA) concentrations (0, 5, 10, or 15 μM) on MS medium, fewer than 0.5 shoots developed per explant. However, when seedling explants were treated with 1.0 μM thidiazuron (TDZ) plus 10 μM BA all cultures produced over 1.0 shoot per culture. Therefore, explant establishment from seedling, mature, or rejuvenated sources was attempted using MS medium supplemented with 10 μM BA plus 0.1 μM TDZ (Finneseth et al., 2000). Seedling explants were about 12 weeks from seeding and had six to ten nodes. Mature explants were taken from new growth forced from excised dormant stems held in a growth chamber. They included stems collected from 26 genetically different mature trees. Rejuvenated explants were from shoots developing on root pieces from mature plants as previously described for root cuttings. Seedling explants established at 100% and developed faster than other explants (Table 2). Explants from rejuvenated stems established at

a slower rate but produced shoots in over 40% of the cultures after 8 weeks. Of the 551 mature explants cultured, 72% were successfully disinfested, but only 4% survived in culture and none produced shoots after 8 weeks in culture. Most mature source explants turned black and lost tissue integrity. Significant browning of the medium was evident in all cultures (Fig. 3). A small number of mature explants continued to survive and produced a limited number of shoot buds after 7 months in culture. However, these never stabilized and no mature cultures survived for more than 12 months in culture.

One accession (A10-11) developed from rejuvenated explant sources showed continued growth and shoot production during subculturing. It has been maintained in culture for over 3 years (Fig. 3D). Tissue culture conditions established for efficient shoot multiplication were MS or Woody Plant Medium (Lloyd and McCown, 1980) with 8.9 μM BA and 2.7 μM naphthalene acetic acid (NAA), 3.0 % sucrose, and 0.7% agar. Cultures were maintained at a photoperiod of 16 h at 20 μmol·s-1·m-2 of light provided by cool white fluorescent bulbs. Culture room temperature was 25 °C.

The ability for single stem explants from 3-year-old cultures of A10-11 to produce shoots on shoot multiplication was investigated using 9.8 μ M IBA plus 5.4 μ M NAA in combination with BA (0 to 20 μ M) (Table 2). Initial explants elongated but did not form additional shoots after 8 weeks in culture (J.N. Egilla, unpublished). These were subcultured to the same medium and after 9 weeks cultures treated with 15 or 20 μ M BA had the greatest number of shoots per culture and 15 μ M BA had the most vigorous shoot growth (Table 2). These data indicate that cultures of pawpaw can retain morphogenetic potential for a considerable time in culture.