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Paul E. Read

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Paul E. Read and Sanjun Gu

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Jim Hruskoci and Paul E. Read

In an effort to increase somaclonal variation in blueberry, a protocol was developed to regenerate viable shoots from internode segments. The explant consisted of the last-formed, fully developed internode taken from 3 different genotypes of Vaccinium grown in vitro. Explants were cultured 6 weeks on Zimmerman's Z-2 medium supplemented with 2iP, zeatin, thidiazuron, kinetin, or BA at concentrations of 5, 25, 50, and 100 uM. Explant response to the treatments varied and included: no response, callus growth only, callus growth and subsequent shoot formation originating from the callus mass, and adventitious shoot formation directly from the internode segment without an intervening callus. Greatest shoot regeneration (20-25 shoots/explant) was obtained on medium supplemented with zeatin at 5, 25, and 50 uM, however treatment response was not consistant across all genotypes. Regenerated shoots could be readily sub-cultured, rooted in soil mix and will be evaluated for somaclonal variation.

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Margaret From and Paul E. Read

Platanthera praeclara, commonly called western prairie fringed orchid, is a showy forb native to seven states and one Canadian province. The species had resisted previous attempts at propagation. Small, isolated populations in the sandhills region of western Nebraska are disjunct and visitation by natural pollen vectors appears to be in decline. Modern cultivation practices and other habitat encroachment factors, including urban development, recreational activities, and natural fluctuations in seasonal water availability all have the potential to exert pressure on current populations. Federal and state permits have allowed a limited hand-pollination study to be conducted on federal land. Hand-pollinated plants showed a greater fruit production compared to control plants receiving no human pollination assistance. Germination studies were conducted using aseptic in vitro techniques. The microscopic seeds possess testa that are extremely hard and resistant to liquid absorption, which presents challenges to germination in vitro. These challenges will be discussed. Alternating cold treatments with room temperatures appeared necessary to promote protocorm development after germination. Three media tested produced varying germination responses. Juvenile plants produced through micropropagation can offer propagules for possible future reintroduction efforts of this protected species.

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Erika Szendrák and Paul E. Read

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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Handan Büyükdemirci and Paul E. Read

Axillary buds of `Valiant' grapevine (Vitis spp.) grown in vitro were transferred onto Murashige and Skoog (MS) medium supplemented with different cytokinin and auxin combinations and concentrations. It was found that culture medium caused statistically important differences in number of nodes, number of fully expanded leaves, number of multiple shoots, number of roots, and length of shoots. MS medium supplemented with 1.0 mg BA/liter in combination with 0.01 mg NAA/L was found to be the best medium for shoot growth and callus production. MS medium supplemented with the combination of 0.5 mg BA/L and 0.01 mg NAA/L was the best medium for explant rooting. The medium containing BA and NAA encouraged better shoot growth than those containing BA alone. When the concentration of BA in the medium was increased, multiple shoot proliferation and teratological structures of explants increased, but the number of small leaves and length of internode decreased. Axillary bud culture led to better shoot growth than was found for shoot apex culture. The presence of leaves positively affected shoot growth from axillary buds. Also placing the axillary buds horizontally onto the medium gave better shoot proliferation and growth than placing them vertically.

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Erika Szendrak and Paul E. Read

The effects of organic compounds most commonly used for orchid micropropagation and the physical condition of the medium were investigated for the development of young temperate orchid protocorms. Separate experiments were conducted with five different temperate orchid species: Dactylorhiza fuchsii, Dactylorhiza maculata, Dactylorhiza majalis, Orchis morio, and Ophrys lutea. Small 2- to 4-mm-wide protocorms were placed in baby food jars (three per jar) containing 50 ml modified FAST medium (Szendrak and R. Eszki, 1993) supplemented with one of eight treatments in a split-plot design with five replications. Both the liquid medium (gyrotary shaker, 125 rpm) and the gelled medium (8 g agar/L) were supplemented with one of the following compounds: 2 g peptone/L; 100 ml coconut water/L; 1 g casein+1 g lactalbumin/L; and 10 g glucose/L as a treatment with a defined compound. All treatments were kept in the dark at 25°C. The number of protocorms/jar were counted weekly over a 6-week-long period and the size and fresh weight of protocorms were measured at the end of the 6th week. In most cases, the liquid medium increased proliferation and the size of the protocorms. However, generally after the 4th week on liquid medium, the development of the protocorms often stopped, but it continued on the gelled medium till the end of the experimental period. The media supplemented with the undefined organic compounds showed a much better effect than the medium supplemented with glucose. Generally peptone and coconut water led to the best development of protocorms, but this varied with species. The development of protocorms into plantlets was normal in all cases.

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Guochen Yang and Paul E. Read

A certain period of cold is needed to break bud dormancy for almost all woody species. A pre-forcing bleach soak has been demonstrated to at least partially replace this requirement (Yang and Read, 1989). Therefore, new softwood growth can be produced in the off-season. Such supple softwood growth is excellent material to be used either as explants for in vitro culture, or as cuttings for macropropagation of woody species. Further studies on pre-forcing bleach soaks were conducted to investigate optimum concentration and duration of soak, and to find the most suitable depth of bleach solution soak, in order to maximize the breaking of bud dormancy. Optimum bud break was obtained by soaking the basal 1/3 of dormant stems in 10% bleach solution for 10 minutes prior to forcing. Soaking dormant woody stems in alcohol solutions prior to placing stems in the forcing solution was also studied. The alcohol soak had negative effects on bud break of spirea, although it showed positive effects for lilac and privet.

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Gouchen Yang and Paul E. Read

BA, IBA and GA3 were incorporated into softwood tissues to be cultured in vitro or rooted as cuttings by adding the plant growth regulators (PGR) at various concentrations to a forcing solution containing 200 mg/l 8-hydroxyquinoline citrate and 2% sucrose. BA and GA3 helped break bud dormancy in autumn-collected stems and increased percent bud-break. IBA inhibited bud break and shoot elongation. Rooting of forced softwood cuttings was enhanced by IBA in the forcing solution, while GA3 inhibited the rooting of plant species tested. When dormant stems were forced with periodic additions of BA (10 mg/l) in the forcing solution, in vitro shoot proliferation was enhanced. However, inclusion of GA3 in the forcing solution reduced shoot proliferation. A pre-forcing NaOCl soak and a pre-forcing treatment with wetting agents accelerated bud break, size and number of shoots available for both micro- and macro-propagation of the woody plant species tested. The forcing solution protocol described is an effective PGR delivery system and it can be used by the propagator to extend the season for obtaining softwood growth suitable for use as in vitro explants or softwood cuttings.