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Paul E. Read

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Paul E. Read and Sanjun Gu

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Guochen Yang and Paul E. Read

A forcing solution containing 200 mg 8-hydroxyquinoline citrate per liter and 2% sucrose has enhanced availability of cutting materials by forcing dormant woody stems in the off-season. Anxins, such as IBA, included in the forcing solution promoted subsequent rooting by increasing root number per cutting and root length for privet. Inclusion of IBA in the forcing solution following the initial use of GA3 in the forcing solution counteracted the undesirable effects of GA3 on rooting and stimulated rooting after taking advantage of the favorable effects of GA3 on bud break and shoot elongation. However, the ability of IBA to counteract the negative effects of GA3 on rooting was dependent on the length of GA3 treatment. The modification of forcing solution system by sequentially including GA3 and then replacing GA3 with IBA expedited propagation of privet. Production of candidate cuttings or explants was stimulated by including GA3 in the forcing solution, and rooting of the cuttings was promoted by subsequent auxin or cytokinin inclusions in the forcing solution to replace GA3 This modified forcing solution system also proved to be a successful and efficient model for propagation of other difficult to propagate woody species.

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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.

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Guochen Yang and Paul E. Read

A forcing solution containing 200 mg 8-hydroxyquinoline citrate per liter and 2% sucrose has been demonstrated to extend the season for obtaining softwood growth suitable for use as explants in micropropagation (Yang & Read 1989). Forcing dormant woody stems in the off-season in this fashion also enhances the macropropagation of woody plant species by providing softwood outgrowth that can be rooted as softwood cuttings. GA3, IBA, IAA and NAA were incorporated into softwood growth which was later used as cuttings for rooting by adding plant growth regulators at various concentrations to the forcing solution. GA3 incorporated into the forcing solution hastened bud break, increased shoot elongation, but inhibited rooting of softwood cuttings taken from stems forced in this manner. IBA, IAA and NAA in the forcing solution exhibited typical auxin effects on rooting of cuttings by increasing root number per cutting and root elongation. In order to expedite macropropagation of woody plants, GA3 and IBA were added SEQUENTIALLY to the forcing solution. Addition of IBA to fresh forcing solution following initial use of GA3 in the forcing solution counteracted the negative effects of GA3 and stimulated rooting. This protocol is proposed as a method to assist propagation in rooting difficult species by softwood cuttings in the off-season.

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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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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.