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Yuexin Wang, Zoran Jeknić, Richard C. Ernst, and Tony H.H. Chen

To improve the efficiency of iris plant regeneration, we tested the influence of several combinations of Kin and NAA in culture media on the induction of morphogenesis and the subsequent development of plantlets. The highest rates of regeneration (67%) were consistently observed in induction media containing 0.5 μm NAA and either 2.5 or 12.5 μm Kin. Developing medium containing 1.25 μm BA was optimal for high regeneration rates and a high percentage of plantlets simultaneously developing shoots and roots. Rooted plantlets were easily acclimatized and transplanted to various soil mixtures, then grown in the greenhouse. Under optimal conditions as many as 8000 plantlets could be regenerated from 1 g of cells in ≈4 months. Chemical names used: kinetin (Kin); 1-naphthaleneacetic acid (NAA); N6-benzyladenine (BA).

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Mahmoud B. Arif and Houchang Khatamian

Friable callus from leaf disks of Rosa hybrida `Tiffany' was initiated within two weeks under dark conditions and 25°C on Murashige and Skoog (MS) medium supplemented with 4 mg.liter-1 2,4-D. Callus was then transferred into MS medium containing 3 mg.liter-1 2,4-D. Within four weeks, rhizogenesis occurred on the callus surface.

The rhizogenic calllus was subculture on MS medium plus 3 mg.liter-1 2,4-D every 4-6 weeks. Within six months from initial culture, somatic embryos were developed on the aging callus in darkness.

Transfer of the aging callus with somatic embryos into 1/2 MS medium containing 1 mg.liter-1 kinetin and maintaining it under 46 μE m-2s-1 light for 16 hrs. resulted in greening of the somatic embryos.

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Mahmoud B. Arif and Houchang Khatamian

Friable callus from leaf disks of Rosa hybrida `Tiffany' was initiated within two weeks under dark conditions and 25°C on Murashige and Skoog (MS) medium supplemented with 4 mg.liter-1 2,4-D. Callus was then transferred into MS medium containing 3 mg.liter-1 2,4-D. Within four weeks, rhizogenesis occurred on the callus surface.

The rhizogenic calllus was subculture on MS medium plus 3 mg.liter-1 2,4-D every 4-6 weeks. Within six months from initial culture, somatic embryos were developed on the aging callus in darkness.

Transfer of the aging callus with somatic embryos into 1/2 MS medium containing 1 mg.liter-1 kinetin and maintaining it under 46 μE m-2s-1 light for 16 hrs. resulted in greening of the somatic embryos.

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Maciej Ladyzynski, Aleksandra Korzeniewska, and Stefan Malepszy

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Nirmal Joshee*, Bipul K. Biswas, and Anand K. Yadav

Centella asiatica L. (Apiaceae family), also called `Indian Pennywort,' is a prostrate, faintly aromatic, and stoloniferous perennial herb with long petiolated leaves. In the Ayurvedic medicine, it is reputed as a nervine tonic along with antibacterial, antifeedant, antileprotic and wound healing properties. Centella contains glycosides, indocentelloside, brahmoside, and asiaticoside. Its leaves are rich in carotenoids and vitamins B and C. In vitro culture techniques which offer a viable tool for mass propagation of plants have recently become increasingly popular for conservation of rare, endangered and threatened medicinal plants germplasm. Centella tissue culture has been reported to experience high incidences of microbial contamination which drastically reduces survival of explants. Thus, the main purpose of this study was to develop an efficient micropropagation technique for Centella asiatica to reduce explant contamination and rapidly disseminate superior clones for research and production. Here we present induction and further development of somatic embryos, using Centella stolons as explants. Somatic embryos were induced in response to 2,4-D shock on MS medium. Initially, somatic embryos appeared as highly nodular callus and eventually developed into somatic embryos that exhibited globular, heart shaped and cotyledonary stages. After auxin shock, cultures were regularly transferred to MS basal medium where somatic embryos completed various developmental stages and then germinated to give rise to new plantlets. In this presentation, we will demonstrate complete protocols for the successful sterilization of Centella explants prepared from plants that had abundance of fungal and bacterial contamination.

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Roberto Botta, Grazia Vergano, Giovanni Me, and Rosalina Vallania

Floral biology of chestnut, from sporogenesis to mature embryo, is described. Microsporogenesis in flowers of unisexual catkins occurred in the first week of June 1991. Anthesis started in mid-June (≈70 days after budbreak) and lasted 2 weeks. In mid-June, in each pistillate flower, six to eight styles began to emerge, and 4 to 7 days later, they were extended fully (i.e., full bloom). In each flower, 10 to 16 anatropous ovules developed from the ovary axis. The megaspore mother cell had formed by the end of bloom. The mature ovule consisted of two integuments and a long, narrow nucellus with a small embryo sac of the Polygonum type. Zygotes were found 15 to 20 days after pistillate flower full bloom. Embryo development followed the Onagrad type, Trifolium variation. Seeds attained full size in mid-September, and fruit were mature in early November. The embryonal axis averaged 4.5 mm long × 2.1 mm wide. An apical meristem and the radicle were evident at opposite ends of the axis.

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R. A. Hoyos and G. L. Hosfield

Opaque globules formed on bean callus induced on primary leaf explants cultured on induction media (IM) containing 10 to 30 mg/l 2,4-D. Calli with globules produce structures reminiscent of somatic embryos (embryoids) after subculture in a liquid challenge medium (LCM). Calli maintained on IM for 2, 3, 4, and 5 weeks produced significantly more (26 to 34/callus) embryoids in LCM than calli maintained on IM for one week (12/callus). Well developed embryoids only occurred after calli were subculture in liquid B5 with 0.1 to 1.0 mg/l IBA. Calli subculture in LCM with > 10 mg/l IBA turned necrotic and died. Embryoids produced in B5 with 2,4-D and NAA (0.1 to 1.0 mg/l) proliferated roots and formed “frosty” appearing structures, respectively. No differences were detected in number or quality of embryoids produced in LCM from callus maintained on IM in continuous light or darkness regardless of the induction time. Ethylene accumulation in IM cultures inhibited globule formation.

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David J. Wolyn and Xiaorong Feng

Asparagus (Asparagus officinalis L.) anthers from flowers of field-grown plants were cultured for five genotypes, four incubation temperatures, and three sampling dates. Treatments were evaluated for total and embryogenic callus production. Incubating anthers at 35C was optimal for initiating embryogenic callus for three genotypes. Another line performed best and equally well at 29 and 32C, while one was recalcitrant to embryogenic callus formation at the temperatures evaluated. For all genotypes, almost half of the anthers produced callus for at least one temperature treatment, hut the percentage of these calli that was embryogenic ranged from 0% to 50%. Sampling date affected response only for specific genotype-temperature combinations. Embryo recovery ranged from six to 14 per callus. For the four responsive genotypes, 77% to 100% of plantlets was haploid. Culturing anthers at several temperatures ranging from 29 to 35C, with repeated samplings of flowers from the field, likely will allow recovery of haploid embryos from many selections. This result will expand the germplasm base to develop all-male asparagus hybrids.

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Don Waneck, H. Mathews, J. Stamp, and R. Bestwick

Zygotic embryo explants of grape cultivar AXR#1 were isolated from maw-e seeds and cultured on medium supplemented with naphthoxy acetic acid beta-(NOA) and benzylaminopurine (BA). Embryo explants dedifferentiated to form embryogenic callus. Globular stage embryos were visible in 9-10 months. On transfer 10 a growth regulator free medium supplemented with charcoal these globular embryos underwent further stages of embryo development. In a period of 30-40 days embryogenic tissues turned into clumps of somatic embryos displaying different stages of development Cotyledonary stage embryos were separated and transferred to basal medium. These embryos developed into complete plants. Cold and desiccation treatment of somatic embryos significantly enhanced the rate of plant conversion. Hypocotyl segments of elongated somatic embryos were good source explant for induction of shoot organogenesis. The hypocotyl-length and the proximity to-shoot-apex were found to influence the rate of shoot induction from hypotyl segments. Multiple shoot complexes which formed on hypocotyl segments were separated and individual shoots were grown on a root induction medium resulting in complete plant development. The possibility of both embryogenic and organogenic modes of plant regeneration make somatic embryos a highly versatile explant source for experiments on genetic manipulation.

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Jane Kahia, Siaka Kone, Lucien Diby, Georges Ngoran, Colombe Dadjo, and Christophe Kouame

pattern which can occur ( Figueira and Janick, 1995 ). Plant regeneration through somatic embryogenesis provides an alternative approach for propagation of plants. Somatic embryogenesis depends on the ability of somatic plant cells to dedifferentiate and