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Abstract

Seed from three octoploid (2n = 8x = 56) Fragaria species [F. × ananassa Duch., F. chiloensis (L.) Duch., and F. virginiana Duch.] and three diploid (2n = 2x = 14) species (F. vesca L., F. viridis Duch., and F. daltoniana J. Gay) were placed in vitro on water agar (WA) and ex vitro on 1 sand : 1 sphagnum (v/v) mix. Seed of the octoploid species germinated best regardless of medium. Octoploid species also exhibited better establishment on modified Boxus proliferation medium than the diploids. Five in vitro germination media were tested for F. vesca and F. × ananassa. Fragaria × ananassa germinated best when WA, or WA + sucrose (Su) + 0.1, or 0.05 strength Boxus proliferation nutrients (Nu) was used as the germination medium. Fragaria vesca showed no difference in germination on Su or Nu. However, establishment of F. vesca on proliferation medium was improved if Nu was included in the germination medium. Data indicated that selection for diploid genotypes with good in vitro establishment is possible.

Open Access

Procedures for in vitro establishment, rapid shoot proliferation, and ex vitro plantlet acclimatization of Cryptocoryne lucens de Witt were determined. Shoot cultures were established from surface-sterilized shoot tips cultured on Linsmaier and Skoog salts and vitamins medium (LS) solidified with 0.8% (w/v) agar and supplemented with 2.0 μm BA and 0.5 μm NAA. The effect of BA (0 to 20 μm) and 0.5 μm NAA on shoot multiplication from single-node and clustered triple-node shoot explants was determined after 35 days. The most efficient shoot proliferation (7.7 shoots/explant) occurred from single-node shoot explants cultured on LS + 20 μm BA and 0.5 μm NAA. Maximum plantlet establishment was achieved by direct sticking of triple-node (cluster) microcuttings in either soilless planting medium or polyurethane foam cubes. Production of highly branched salable plants from microcuttings was possible within 18 weeks. Chemical names used: N-(phenylmethyl) -1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA).

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Weigela Thunb. consists of 12 species distributed throughout Northeast Asia. Diervilla Mill. is a closely related genus containing three species endemic to North America. Taxa from both of these genera are important nursery crops. Hybrids between these genera could potentially combine the excellent cold hardiness and adaptability of Diervilla with diverse forms, foliage colors, and flower colors found in Weigela. Prior attempts to create intergeneric hybrids between these genera were unsuccessful and resulted in embryo abortion before seeds matured. To overcome this barrier, ovule culture and micropropagation procedures were used to develop intergeneric hybrids. Cleaved amplified polymorphic sequences (CAPS) analysis was used to verify hybrids. Intergeneric crosses, D. lonicera × W. middendorfiana, D. sessilifolia × W. florida (two clones), and D. lonicera × W. florida were attempted. Crosses of D. lonicera × W. middendorfiana did not produce viable hybrids. From the remaining three crosses, a total of 544 plants were obtained from 1278 ovules. About 85% of the 544 plants appeared very chlorotic or had low vigor, and senesced when transferred to multiplication medium. Only 80 of the 544 plants were successfully maintained in tissue culture, of which 10 have been successfully transferred ex vitro. CAPS analysis indicated that a majority of these plants were hybrids. Further studies are focused on improving tissue culture procedures and other methods to develop tetraploids to increase plantlet vigor and fertility.

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In vitro shoot cultures for two birch species, Asian white birch (Betula platyphylla) and paper birch (Betula papyrifera), were initiated from shoot tips of mature trees and maintained in MS (Murashige and Skoog) medium containing 3% sucrose and 5–10 μM (micromolar) benzyladenine (BA). The effect of such factors as genotype, basal medium, and plant growth regulator (PGR) on proliferation was investigated. Shoots were proliferated in both MS and woody plant medium (WPM) supplemented with different concentrations of thidiazuron (TDZ), BA, and kinetin (Kin). Two birch species responded differently to these factors. In general, more shoots were proliferated in WPM than in MS medium. The maximum proliferation rate of Asian white birch was achieved by being cultured in WPM containing 4–8 μM TDZ, while paper birch gave rise to the maximum proliferation rate in WPM supplemented with 20 μM BA. Interactions between genotype and medium or cytokinin were found. Shoots produced on media with TDZ had thick stems and small, dark green leaves. Microshoots can be rooted both in vitro and ex vitro with or without IBA treatment. Plants were regenerated from leaf tissues of Asian white birch. Adventitious shoots regenerated when in vitro leaves were cultured on WPM supplemented with 10–20 μM BA with 2-week dark treatment. The effect of genotype, PGR, and culture condition on in vitro regeneration of birch species is being tested.

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Callus was initiated from leaves of Gladiolus cv. `Balady' on MS medium containing 1.0 mg/L NAA, 0.1 mg/L 2,4-D, and 0.5 mg/L kinetin. Organogenesis from callus was induced on medium containing 0.5, 1.0, 1.5, or 2.0 mg/L of either BA, kinetin, or TDZ. TDZ was more effective and resulted in a higher percentage regeneration and regenerant number. The microshoots produced were then propagated in vitro and cormel production was studied. Maximum shoot number (25.1) was obtained on medium containing 1.0 mg/L TDZ without auxin supplements in liquid shaking culture. In vitro cormel formation was significantly enhanced by B-9 and paclobutrazol. Increased sucrose concentration (4% to 5%) proved the most effective for cormel formation. Optimal dormancy break was obtained by storing cormels at 5°C for 1 month or by soaking them for 5 sec with 50 mg/L GA3. In-vitro rooting was achieved on solid medium containing NAA, IAA, or IBA, with higher root number recorded on NAA-treated cultures. Rooted microshoots were successfully acclimatized for ex vitro conditions and grown in the greenhouse. Plants produced from in-vitro propagation showed similar morphological characteristics of plants propagated by direct corm planting in the greenhouse.

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Achimenes is a summer-flowering pot plant commonly propagated by shoot tip cuttings taken from rhizomes released from dormancy. Micropropagation was used in this study in order to establish a protocol for producing plants in winter when Achimenes are not usually available. Leaf segments, taken in August 1993, from hybrids `Flamenco', `Rosenelfe', `Bella', and `Sandra' grown in a greenhouse, were cultured on a modified Murashige and Skoog (MS) medium supplemented with 0.1 mg·liter–1 BA and 0.5 mg·liter–1; shoots proliferated without callus formation. Leaf explants taken from the proliferated shoots were placed on MS medium with 0.5 mg·liter–1 BA and 0.1 mg·liter–1 NAA for 8 weeks for further shoots proliferation. `Bella' showed vigorous growth and produced the most shoots (82) with no rhizomes, whereas `Flamenco' had the least shoots (28) along with rhizomes. Shoot tips were then transferred on MS medium supplemented with 0.5 mg·liter–1 NAA for 6 weeks where more vigorous shoots developed along with roots. Microcuttings were directly stuck ex vitro under moisture and rooted well in 4 weeks before planting in individual culture and flowered normally. These results provide the basis for a successful production of Achimenes hybrids for growth and flowering in winter months provided optimal temperature and irradiance levels are given.

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The temperate native terrestrial orchids are endangered species. Their propagation from seeds poses specific problems. It is well known that orchid seeds are devoid of endosperm and in nature they need microscopic fungi in a symbiotic relationship for germination. We developed a successful asymbiotic in vitro culture method for germinating seeds of several temperate orchid species and for maintaining the cultures of young plantlets. The medium used for both germination and seedling culture was a modified FAST medium. Seeds were surface-disinfested for 10 minutes in a 10% calcium hypochlorite solution. After sowing, the cultures were kept under dark condition at 10–12°C for 4 weeks. After that the cultures remained in the dark, but the temperature was raised to 25–26°C until germination occurred. Thereafter cultures required alternating seasonal temperatures: 25–26°C from the beginning of April to the end of September and 17–19°C from October to March. For the development of the young plantlets natural dispersed light and prevailing day-length was favorable. After 2 years of aseptic culture they were suitable for transfer ex vitro. Different stages of seed germination and plant development were observed using a scanning electron microscope and will be included in this presentation. Further observation of the effects of different environmental factors is currently under investigation.

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Cultures of several orchid species [Barlia robertiana (Loisel.), Dactylorhiza fúchsii Soó, D. incarnata (L.) Soó, D. maculata (L.) Soó, D. majalis (Rchb.), D. saccifera (Brong) Soó, D. sambucina (L.) Soó, Gymnadenia conopsea (L.) R.Br., Himantoglossurn hircinum (L.) Spreng., Ophris sphegodes Mill., Orchis coriophora ssp. fragrans L., Orchis laxiflora ssp. palustris Lam., Orchis mascula L., Orchis morio L., Platanthera bifolia (L.) Rich., Spiranthes aestivalis (Poir.) Rich.] were initiated with fresh ripe seeds from desiccated fruit and 4-month-old in vitro seedlings. The medium used for both germination and seedling culture was a modified FAST medium. Samples for the scanning electron microscope (SEM) surveys were taken from the in vitro cultures and some plant materials were collected from their native habit. Samples were observed with a Tesla BS 300 SEM. Seeds ranged from 300 to 450 μm in length and were flask-shaped. The first germination step is opening of the seedcoat, when the first few white cells will be visible. After a few weeks, the apical meristem appears. The young protocorm is covered with numerous translucent rhizoids. In the last stage of germination, the first root and the first true leaf start to develop. After 2 years, they are suitable for transfer ex vitro. Structure of the mature organs and tissues can be examined at this stage.

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Meristem culture and/or thermotherapy were used to eliminate viruses from ornamental Dianthus gratianopolitanus Vill. (`Spotti' and `Frosty Fire') mother plants. Shoot tip, leaf, node, and ovary explants collected from greenhouse-maintained, virus-free plants were cultured in vitro for shoot initiation on Murashige and Skoog (MS) medium containing BAP, kinetin, or 2-iP with or without IAA or NAA. Culture of shoot tips in MS with 0.57 μm IAA and node explants in MS with 2.46 μm 2-iP is recommended for `Spotti' cultivar. In `Frosty Fire', optimum number of axillary shoots was obtained from shoot tip and node explants in MS without plant regulators. Leaves and ovaries were not adequate explants for D. gratianopolitanus micropropagation because none or only a low percentage of explants regenerated shoots. High levels of cytokinins increased the number of shoots per explant but also increased the production of aberrant phenotypes and induced hyperhydricity. Adventitious shoots rooted in vitro with auxins, but maximum rooting was 97% ex vitro without auxins. This study demonstrated that D. gratianopolitanus can be successfully micropropagated. Chemical names used: 6-benzyladenine (BAP); kinetin (KIN); 6-(γ,γ-dimethylallylamino)-purine (2iP); indole-acetic acid (IAA); indole-3-butyric acid (IBA); α-naphthaleneacetic acid (NAA); gibberellic acid (GA3).

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Meristem culture and/or thermotherapy were used for virus elimination from ornamental Phlox paniculata L. (`Blue Boy', `Orange perfection' and `Starfire') mother plants. Shoot tip, leaf, node and flower ovary explants collected from greenhouse-maintained virus free plants were cultured in vitro for shoot initiation. Adventitious shoot initiation was observed on Murashige and Skoog (MS) medium containing the cytokinin BA with or without the auxin NAA. The addition of 0.4 mg·L-1 thiamine, 0.4 mg·L-1 folic acid, and 40 mg·L-1 adenine sulfate to the MS medium did not improve the regeneration rate. Multiplication and rooting were genotype dependent. Blue Boy and Orange Perfection cultivars regenerated the maximum number of shoots from leaf explants. `Blue Boy' leaf explants from in vitro plants had a lower regeneration rate than explants from greenhouse plants. Cultivar `Starfire' had the highest shoot formation with open flower ovary explants and failed to regenerate from leaf explants. In vitro rooting of adventitious shoots in the presence of auxins (IAA, NAA, or IBA) with or without BA was less effective than ex vitro rooting. Chemical names used: 6-benzyladenine (BA); indole-acetic acid (IAA); indole-3-butyric acid (IBA); α-naphthaleneacetic acid (NAA).

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