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Michael E. Compton

Several methods have been published on shoot regeneration from watermelon cotyledon explants. The major differences in regeneration protocols include the light environment in which seeds are germinated and the cotyledon region used. The purpose of these experiments was to compare the two main protocols for plant regeneration and develop one general procedure. To fulfill this objective, seeds were germinated in vitro in darkness or 16-hr light photoperiod for 7 days. Cotyledon explants from four watermelon cultivars (`Crimson Sweet', `Minilee', `Sweet Gem', and `Yellow Doll') were prepared from both dark- and light-grown seedlings. Apical and basal halves were obtained by making a cut across the cotyledon width. Apical and basal quarters were made, for comparison, by cutting apical and basal halves longitudinally. All explants were incubated on shoot regeneration medium for 6 weeks followed by a 3-week cycle on shoot elonga-tion medium. The percentage of cotyledons with shoots was 1.7-fold greater for cotyledons derived from seedings incubated in darkness than those germinated in light. Shoot formation was about 10-fold greater for explants from cotyledon basal halves and quarters than apical halves and quarters. According to these results, the best watermelon regeneration protocol should consists of basal explants from in vitro-germinated seedlings incubated in the dark for 7 days.

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Michael J. Tanabe and Nicole Wakida

Noni, Morinda citrifolia, is receiving a lot of attention for its potential medicinal effects. Hawaii is an ideal growing environment for this plant, where it has been used for many purposes, including medicinal ones, by ancient Polynesians. Currently, there is a rapidly developing noni industry in the state of Hawaii. Propagation of this plant is almost exclusively by seeds, and germination generally requires a couple of months without preconditioning or about a month if mechanically scarified. We developed an in vitro protocol that significantly improves percent germination rate by altering incubation temperature and the in vitro culture basal medium. Germination time was decreased to 4 days when the embryo was extracted and exposed to 31 °C. A basal medium containing 1/2 Murashige and Skoog (M&S) salts was the most effective in reducing germination time and increasing percent germination. Stem pieces obtained from in vitro-propagated seedlings produced callus when explanted in 1/2 M&S containing various levels of naphthalene acetic acid (NAA). The most effective treatment was 0.5 μm NAA and the least effective treatment was 2 μm NAA. Treatments without NAA did not produce callus. Calli treated with 4.40 μm 6-benzylaminopurine (BA) or 8.80 μm BA were the most effective in promoting caulogenesis. We also demonstrated that the number of first generation seedlings produced from each embryo could be increased by treatment with 8.80 μm BA.

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I. David van der Walt and Gail M. Littlejohn

This paper is a revision of a chapter of a MS thesis submitted by I.D. van der Walt to satisfy the requirements in plant breeding at the Univ. of Stellenbosch. We thank Frikkie Calitz and Marieta van der Rijst for their assistance in statistical

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Hector G. Nunez-Palenius, Daniel J. Cantliffe, Harry J. Klee, and Don J. Huber

Pollen germination timing has a paramount role in fertilization of a flower. Rapid germination and outgrowth of a pollen tube that penetrates the stigma is required. Physical and biological factors can affect pollen germination timing. The objective of this study was to determine if ACC oxidase antisense gene expression could influence in vitro pollen germination and in vitro pollen tube length growth. A transgenic (ACC oxidase antisense) `Galia' male parental line had a reduced fruit set compared to its wild type. Likewise, embryo abortion and empty seeds after self-pollination in a `Galia' male parental line were observed. Wild type and transgenic `Galia' male parental line melon plants were grown in a greenhouse according to the practices of Rodriguez (2003). Male flowers were collected from these plants between 10 to 12 am; pollen was obtained by dipping the anther in germination medium (10.25% sucrose, 0.031% calcium nitrate, 0.015% boric acid, 0.0075% KNO3, and 0.016% MgSO4) at 25 °C and analyzed immediately, either for total percentage of germination after 5 minutes of incubation or to measure pollen tube growth rate every 5 minutes during 1 hour. Each flower provided an average of 250 pollen grains. Assays were conducted by using the “Hanging Drop Method” (Okay and Ayfer, 1994). Percentage of pollen germination in WT `Galia' male parental line was greater than the transgenic line. Likewise, in vitro pollen tube growth in wild type `Galia' melon was greater than pollen from the transgenic line. Possibly the ACC oxidase antisense gene expression in `Galia' male parental line may have had an influence on the reduced fruit set observed.

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Vladimir Orbović, Manjul Dutt, and Jude W. Grosser

decrease of abscisic acid (ABA) levels in seeds ( Farnsworth, 2000 ). Thus, our data suggest that the change in ABA levels in older seeds did not affect their subsequent germination in vitro. Another possibility is that ABA levels did not change in seeds

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Vital Hagenimana, Ronald E. Simard, and Louis-P. Vézina

Coopération Institutionnelle Laval/UNR financed by Canadian International Development Agency. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked

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Erika Szendrák, Paul E. Read, Eszter R. Eszéki, Elizabeth Jámbor-Benczúi, and Aniko Csillag

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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Yousef I. Dlaigan, A.E. Said, and M.A. El-Hamady

Several trials were conducted with the objective of obtaining an explant for the establishment of date palm root culture in vitro. These trials included disinfecting and germinating seeds of three cultivars on several autoclaved culture media, the influence of incubation temperature on different germination parameters, and the quality of roots before excision and after culture in nutrient media. Three culture media were used: distilled water only; minimal organics that consisted of MS salts, 3% sucrose, modified White's organics, 0.01% inositol, and 0.15% activated charcoal; and 1/2 MS salts mixture, 3% sucrose, and 1/2 modified White's organics. All three media were solidified with 0.7% agar. The seeds were incubated at 25 or 35C for germination. The study revealed the difficulty of seed disinfection. We immersed seeds in 20% to 40% Clorox, with two to four drops of Tween-20, for 30 to 60 minutes and then rinsed them four to five times in deionized distilled water before culturing. The minimal organics medium supported optimal growth of excised roots, and incubation at 35C significantly improved germination. The use of 10-mm-long root tips as explants for culture initiation gave the best growth and elongation. In addition, the growth and elongation of excised root tips increased significantly as the distance from it to the apex of the cotyledonary sheath increased.

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Chad Finn, Kirsten Wennstrom, and Kim Hummer

Populations of 40 Rubus sp., representing the Malachobatus, Idaeobatus, Eubatus, and Anoplobatus, were planted in the field in 1994. To get a preliminary idea of how successful crosses between these species and standard cultivars would be, 58 crosses were attempted between standard cultivars and randomly selected genotypes of the 14 species that produced a significant number of flowers in 1995. Diploid species were crossed with `Tulameen' and `Meeker' raspberry and the tetraploid species with `Cherokee' and `Chester' blackberry. Twenty-two crosses produced seed lots ranging from 8 to 630 seeds. Crosses were successful with R. caesius, R. caucasicus, R. coreanus, R. georgicus, R. parvifolius, R. rosifolius, and R. sumatranus. Crosses were not successful with R. eustephanos, R. insularis, R. innominatus, R. lambertianus, R. sachalinensis, R. setchuenensis, R. swinhoei, and R. tsangorum. In vitro seed germination was attempted with all crosses. Larger seed lots were also germinated using standard procedures for Rubus. There is a great deal of variability in leaf morphology of the young seedlings within a cross that suggests that some or all of the seedlings are true hybrids. Seedlings that are not true hybrids could result from contaminant pollen or, as in R. armeniacus, pseudogamous embryo formation. Crossing results from 1995 and 1996, including crosses attempted and seed numbers per cross, will be presented along with, for the 1995 crosses, the number of germinated seedlings and our assessment of whether they appear to be true hybrids.

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J.C. Vlahos and M. Dragassaki

Ebenus cretica, Leguminosae, is a characteristic endemic plant of the Mediterranean island of Crete. It is a perennial bush up to 1 m tall with composite pubescent leaves and pinky red or purple flowers on 5- to 20-cm-long racemes. The fruit is surrounded by the calyx and contains one seed. The plants grow on rocky hillsides in alkaline soils at an altitude of up to 600 m and flower from April to June. Ebenus has the potential for use as a container or landscape flowering plant, and this study was aimed at finding methods to propagate it either by seed or by shoot cuttings. Seed collected from native plants in late July/Aug. 1992 germinated well (70% to 90%) without scarification in a commercial potting mix. Fifty percent of the seed germinated in vitro between 13 and 25 days, depending on temperature and substrate used. Temperatures of 25 or 30C in light at a pH ≈6.0 favored germination. Removal of the dry calyx coating the seed enhanced germination and emergence. For rooting Ebenus cuttings, several concentrations of IAA, IBA, and NAA were used in combination with different types of cuttings (soft or hardwood, tip or basal, cultivated or wild). Best results were obtained by wounding the base and dipping shoot-tip cuttings (12 cm long) in 600 mg IBA/liter for 16 hours. Significant differences, however, were observed among germination and rooting percentages when seeds or cuttings were taken from different plants due to genetic diversity. Therefore, selection is required for optimal results.