Japanese chestnut (Castanea crenata Sieb. et Zucc.) was micropropagated from nodal explants of 2-month-old seedlings, and the regenerated plantlets were acclimatized after potting. For in vitro establishment, 5 μm zeatin was more effective than 5 μm BA or TDZ, and Sato's (BW) medium was as effective as MS(½NO3) medium. Driver-Kuniyuki walnut medium produced more shoots showing hyperhydricity symptoms. In the multiplication culture, the higher the concentration of zeatin added to BW medium, the greater the number of shoots showing hyperhydricity, although the longest shoot was obtained when 6.9 μm zeatin was added. After planting in ½BW medium with 15 μm IBA for 5 d to induce rooting, shoots planted in ½BW medium plus vermiculite gelled with Gellan Gum (SV substrate) rooted better than those in either the gelled medium without vermiculite (S substrate) or vermiculite plus liquid medium (V substrate). One third of the shoots planted in the V substrate died, although 62% of the surviving shoots rooted well. Of the shoots planted in the S substrate, 83% survived, but only 35% of these survived rooting. Shoots rooted in the SV and V substrates survived well after the completion of acclimatization, and shoots rooted in the SV substrate grew more vigorously after potting. Chemical names used: 6-benzyladenine (BA); 6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin); indole-3-butyric acid (IBA); 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ).
Takuya Tetsumura and Kensuke Yamashita
Takuya Tetsumura and Hisajiro Yukinaga
Microshoots of Japanese persimmon (Diospyros kaki L. cv. Jiro) were rooted in vitro. The roots were excised and cultured on solidified Murashige and Skoog medium. After 20 days of culture, adventitious shoots formed spontaneously and directly from the roots. Of all the tested cytokinins, 10–5 m zeatin in combination with 10–8 m IAA was the most effective in stimulating production of adventitious shoots. CPPU and 2iP also were effective cytokinins. Addition of a high concentration of auxin, especially 2,4-D, to the medium inhibited adventitious shoot formation. The percentage of root segments forming adventitious shoots increased with increasing segment length. Almost all of the longest roots (4 to 6 cm) formed adventitious shoots. Chemical names used: 6-benzyladenine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); indole-3-acetic acid (IAA); 2-isopentenyladenine (2iP); N-phenyl-N′-(2-chloro-4-pyridyl)urea (CPPU).
Takuya Tetsumura and Hisajiro Yukinaga
When cultured in vitro, roots of four Japanese persimmon (Diospyros kaki L.) cultivars formed adventitious shoots on MS medium with 10 μm zeatin and 0.01 μm indole-3-acetic acid, although their organogenetic capacities varied. Histological study revealed that the origin of the adventitious shoots was the pericycle. The regenerated shoots grew well on the shoot proliferation medium (MS with 5 μm zeatin). Final rooting percentages of shoots regenerated from roots of three of the four cultivars were greater than those of shoots that originated from shoot tips and that had been subcultured >50 times. Shoots regenerated from `Jiro' roots rooted 10 days earlier, had more roots than those from shoot tips, and maintained higher rooting ability over ten subcultures. Rooted `Hiratanenashi' shoots regenerated from roots survived better after acclimatization than those from shoot tips. No obvious variants were observed either in vitro or in the field. The trees regenerated from roots flowered within 4 years. These findings suggest that partial rather than true rejuvenation was responsible for both the early flowering and the juvenile characteristics, i.e., the enhanced rooting ability, observed in the regenerated plants. Chemical name used: 6-(4-hydroxy-3-methylbut-2-enylamino) purine (zeatin).
Takuya Tetsumura, Hisajiro Yukinaga and Ryutaro Tao
Growth of micropropagated Japanese persimmon trees (Diospyros kaki L. cv. Nishimurawase) during the initial 3 years after field establishment was compared with that of grafted trees on seedling stocks. Judging from the mean length of annual shoots per tree and the yearly increases in height, trunk diameter, and top and root dry mass, the grafted trees on seedling stocks grew poorly during the first and second growing seasons, while micropropagated trees, raised in an outdoor nursery, developed poorly only during the first growing season. In contrast, micropropagated trees raised in pots fared well soon after field establishment. These trees had more fine than middle and large roots; in contrast, grafted trees on seedling stocks had one large taproot, which died back to some extent after field establishment, with few fine roots.
Takuya Tetsumura, Ryutaro Tao and Akira Sugiura
A potentially dwarfing rootstock for japanese persimmon (Diospyros kaki L.) was propagated by single-node stem cuttings taken from root suckers. When a mature tree was cut down at ground level and part of the roots was exposed to the air, numerous suckers formed on the exposed parts of the roots. Single-node stem cuttings 3 to 4 cm (1.2 to 1.6 inches) long survived and rooted better than 10-cm (3.9-inch) and 25-cm (9.8-inch) leafy stem cuttings with several buds. Dipping cuttings in 3000 mg·L-1 (ppm) IBA for 5 s or in 25 mg·L-1 IBA for 24 h resulted in similar rooting. Most of the single-node stem cuttings taken in late-June and July survived and rooted well, whereas those prepared in late August rooted poorly and few survived. The survival and rooting percentages were unaffected by the position on the suckers (top vs. base) from which cuttings were taken. High relativehumidity in the propagation frame appeared to enhance survival and rooting. This clonal propagation method will make a rapid multiplication of japanese persimmon, a difficult-to-root species, possible. Chemical name used: indole-3-butyric acid (IBA).
Hirotoshi Tsuda, Hisato Kunitake, Yo Aoki, Akiko Oyama, Takuya Tetsumura, Haruki Komatsu and Katsunori Yoshioka
We tested efficient in vitro methods for screening the genotypes with higher pH tolerance using multiple shoots of intersectional hybrids between Vaccinium corymbosum ‘Spartan’ and V. bracteatum. The response of the four hybrid clones tested to different pH levels was clone-dependent in vitro. An apparent difference was found in the rooting rate among the hybrid clones even at higher pH levels; the rooting rates of JM4 (91%) at pH 8.0 indicated a significantly high value compared with other clones (JM1: 24%, JM2: 9%, JM3: 8%, ‘Spartan’: 0%). Furthermore, JM4 showed constantly high rooting rates (91% to 100%) at all pH levels with no significant differences. Similar differences in the root characters of the hybrids were also confirmed by checking the viability of roots using fluorescein diacetate (FDA)/propidium iodide (PI) staining after dipping the roots of in vitro-produced shoots in liquid medium at different pH levels for 6 hours. These results suggest that an in vitro screening method using the rooting rate of multiple shoots and the viability test of roots by FDA/PI staining as a marker could become a very useful tool for the selection of germplasm with tolerance to higher pH within a short time using small planting spaces. In addition, JM4, which showed a high rooting rate at pH 8.0, could be useful in breeding new cultivars with higher pH tolerance.