In Vitro Systems for Propagation and Improvement of Tropical Fruits and Palms

Persimmons belong to the genus Diospyros in the family Ebenaceae from which comes the ebony wood, a valuable timber for furniture. The genus contains nearly 400 species, most of which are natives of tropical and subtropical regions. Among them, only four species are important for their fruit production. They are D. kaki L., D. lotus L., D. virginiana L., and D. oleifera Cheng (Kitagawa and Glucina 1984). The most important species is undoubtedly D. kaki, Japanese or Oriental persimmon, often referred to simply as Kaki (Fig. 1). D. lotus, the Asian date plum, is used as a fruit in Asia and also as a rootstock for Japanese persimmon. D. oleifera is grown in China mainly as a source of tannin. D. virginiana, a native of North America, grows wild but is seldom grown commercially.

Micropropagation of Japanese persimmon (Diospyros kaki L.)

In this works, a simple, efficient and repeatable protocol was developed for in vitro regeneration via callus-mediated organogenesis of Neolamarkia Cadamba using cotyledonary petioles and hypocotyls. Effects of basal medium, plant growth regulators, the types and age of explant on the formation of adventitious buds/shoots were studied. Meanwhile, histological analysis for early ontogenic stages and genetic stability assessment by flow cytometry were investigated. Our investigation demonstrated that, compared with 6-benzyladenine (BA), N6-(2-isopentenyl) adenine (2-ip), Thidiazuron (TDZ) was the optimal cytokinin for buds/shoots induction on cotyledon and hypocotyl explants. Douglas-fir and sugar pine medium (DCR) supplemented with 22.7 μM TDZ and 0.27 μM α-naphthalene acetic acid (NAA) was most effective on bud induction, with the highest bud-induction rate and numbers of buds on cotyledon and hypocotyl explants. The available shoot per explant hit 35.2 when the induced callus sub-cultured to a medium without TDZ. It was found that TDZ could promote induction of the callus and the buds, however, continuous exposure beyond 4 weeks of supplemented high concentration (exceed 11.35 μM), TDZ was harmful to the proliferation and growth of buds/shoots. DCR appeared more efficiency than Murashige and Skoog medium (MS), Woody Plant medium (WPM), anther culture of cereal crops medium (N6) on bud induction. Age of cotyledon and hypocotyl explants in 20-day to 25-day was most beneficial to adventitious buds/shoots formation. Histological investigation confirmed that the buds originated from the wounded incisions of cotyledonary petiole and hypocotyl fragments, with callus formation. The regeneration plantlets were successfully acclimatized in greenhouse, yielded above 95% survival rate in field, exhibited normal morphology and growth characteristics. The analysis of flow cytometry on N. cadamba indicated no variation in the ploidy levels between the regenerated plantlets and the donor trees. The developed procedure can be used for mass production, germplasm exchange and transgenic studies to improve the resistance of the species via Agrobacterium-mediated.

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High frequency regeneration of plants via callus-mediated organogenesis from cotyledon and hypocotyl cultures in a multipurpose tropical tree (Neolamarkia Cadamba).

Neolamarckia cadamba is a fast-growing and deciduous tropical hardwood with anatomical, morphological, and chemical characteristics that make it suitable for building materials, pulp production, and m

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Direct adventitious shoot organogenesis and plant regeneration from cotyledon explants in Neolamarckia cadamba

Using 7 Japanese persimmon cultivars (‘Hiratanenashi’, ‘Jiro’, ‘Hana-gosho’, ‘Aizumishirazu’, ‘E-gosho’, ‘Kurogaki’ and 2 strains, normal and dwarf, of ‘Nishimura-wase’), optimal micropropagational conditions for culture establishment, shoot proliferation and rooting were investigated.1. For culture establishment, both the types (N6-Benzyladenine: BA or N6-Isopentenyladenine: 2iP) and concentrations (1_??_20mg•l-1) of cytokinin were tested with dormant bud explants of ‘Jiro’ and ‘Kurogaki’ on modified MS medium with nitrates reduced to half strength (MS(1/2 NO3)). Optimum concentrations of BA and 2iP for ‘Jiro’ were 5mg•l-1 and 5_??_20mg•l-1, respectively, and those for ‘Kurogaki’, 2_??_5mg•l-1 and 5-20mg•l-1. Explants of ‘Jiro’ produced more shoots than those of ‘Kurogaki’ during this stage and were established more easily. Other cultivars except ‘Hana-gosho’ could be established readily on the MS (1/2 NO3)medium supplemented with 5mg•l-1 BA.2. For shoot proliferation and elongation also, both the types (BA or 2iP) and concentrations (1_??_20mg•l-1) of cytokinin were tested with ‘Jiro’, ‘Aizumishirazu’, ‘E-gosho’, ‘Kurogaki’ and 2 strains of ‘Nishimura-wase’ explants which had been established and subcultured on MS (1/2 NO3) medium supplemented with 5mg•l-1 BA. BA was more effective for shoot proliferation, while 2iP was better for shoot elongation. In most cases, optimum concentration of each cytokinin was 5mg•l-1. The dwarf strain of ‘Nishimura-wase’ was more responsive to either cytokinin than the normal strain.3. For rooting, periods of dark treatment (0_??_15 days) and 3-indolebutyric acid (IBA) concentrations (0_??_1000mg•l-1)for acute exposure were tested with ‘Hiratanenashi’ shoots which had been elongated at 5mg•l-1 2iP in the MS (1/2 NO3) medium. More than 6 days of darkness enhanced rooting, with the highest rooting percent being 9 days of treatment. Optimum IBA concentration for rooting was 250mg•l-1. Shoot cuttings of 5 cvs, ‘Hiratanenashi’, ‘Jiro’, ‘Aizumishirazu’, ‘E-gosho’ and ‘Kurogaki’ rooted with final rootings of 96%, 80%, 74%, 44% and 8%, respectively, by this best combination of darkness and IBA treatment.

In Vitro Shoot Proliferation and Rooting of Several Japanese Persimmon Cultivars

Cambial tissues from the twigs of a mature Japanese persimmon, Diospyros kaki Thunb, cv. ′Tsurunoko′, were used as the starting material in this experiment. Callli were induced on various callus induction media. Root formation in the calli was observed on Murashige and Skoog′s medium containing 1ppm NAA. KIN in combination with NAA did not inhibit to the root formation at 1ppm. Root formation in the callus depended largely on the type of medium on which the callus was isolated and subcultured and the number of successive subcultures on a given medium. When the callus was isolated on Murashige and Skoog′s medium containing NAA and KIN, roots were formed in the first passage, whereas when the callus was isolated on Murashige and Skoog′s medium containing 0.1% yeast extract or 0.1% phytone, NAA, and KIN, the first root formation was not observed until the callus was subcultured on the medium several times.Roots were never formed in calli which were isolated and subcultured on Murashige and Skoog′s medium containing 2, 4-D or IBA instead of NAA, nor were either Wolter and Skoog′s medium or a modified Wolter and Skoog′s medium containing NAA and KIN suitable for root formation.Bud initials were formed in callus which was subcultured several times on Mura shige and Skoog′s medium containing NAA and KIN. KIN was effective in promoting bud initiation in combination with NAA. These initials turned green under continuous light conditions, but they did not succeed in developing themselves into plantlets on various media. Abnormal structures were initiated in callus cultured on Murashige and Skoog′s medium containing NAA and KIN, but these structures were not identifiable as either root or bud initials.

The Induction and Formation of Organs in Callus Cultures from Twigs of Mature Japanese Persimmon (Diospyros kaki Thunb.)

Multiple meristematic nodules were induced on leaf segments from the Japanese persimmon (Diospyros kaki Thunb.) ‘Fuyu’ that were cultured on a solidified half-strength Murashige and Skoog’s medium containing 2% (w/v) sucrose and 1 mM thidiazuron (TDZ). The nodules 1 to 3 mm in diameter were formed in the cut ends and abaxial side of the leaf segments two weeks after inoculation. The nodule changed from pale green to dense green and multiplied to form many daughter nodules. Adventitious buds were thereafter formed on the nodules, suggesting that the meristematic nodule is an intermediate structure for bud differentiation. The adventitious buds grew poorly on the medium with TDZ, but resumed growth and developed into shoots after transfer to the medium containing 2% (w/v) sucrose and 10 mM zeatin. The meristematic nodules survived more than one year on the medium containing 2% (w/v) sucrose and 1 mM TDZ without transfer, and did not lose their ability to form adventitious buds for more than 5 years when transferred regularly to a fresh medium. These results suggest that the meristematic nodule is a promising material for propagation and long-term conservation of this plant.

Tomosaburo Yokoyama

Papers

The Induction and Formation of Organs in Callus Cultures from Twigs of Mature Japanese Persimmon (Diospyros kaki Thunb.)

Adventitious bud formation through nodule induction by thidiazuron in cultured leaf segments of the Japanese persimmon (Diospyros kaki Thunb.)

Relationship between Ontogenic Age and Formation of Bud from Leaf Segments in Japanese Persimmon, Diospyros kaki, THUNB.