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Theophilus Asante* and Guochen Yang

The role of plant growth regulators in enhancing axillary shoot proliferation, callus production and root initiation of the American chestnut (Castanea dentata) was investigated. Micropropagated shoots were used as explant materials. Different concentrations of indoleburytic acid (IBA), kinetin, N-(2-chloro-4-pyridyl)-N'-pheylurea (CPPU), naphtha-leneacetic acid (NAA), thidiazuron (TDZ), zeatin, 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were added to McCown woody plant medium (WPM) to evaluate their effects on in vitro shoot proliferation, callus production, and root initiation. Differences in the number of shoots (primordia), morphology of micropropagated shoots, and amount of callus were observed between the plant growth regulator treatments and concentrations. Explants cultured in media containing CPPU or TDZ produced more shoots (primordia). Callus production increased from NAA to IBA to 2,4-D to 2,4,5-T.

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Guochen Yang and Marihelen Kamp-Glass

An efficient and reliable protocol of in vitro shoot regeneration must be first established to have a successful genetic transformation. As a member of legume family, alfalfa is very difficult for direct shoot regeneration. There is no published information on direct shoot organogenesis, although success has been well documented on embryogenesis, which must go through callus stage. Different plant growth regulators at various concentrations were evaluated for callus initiation, development, and direct shoot regeneration. Multiple shoots were produced directly from each individual explant. This will provide an efficient means for production of transgenic alfalfa plants. Therefore, genetic transformation of Medicago germplasm will be significantly expedited.

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Guochen Yang and Marihelen Kamp-Glass

Multiple shoots were produced directly from each explant. BA, zeatin, or TDZ were evaluated on callus initiation, development, and shoot organogenesis. Callus production was promoted when BA or zeatin was added in culture medium. However, no shoots were produced from such calli. Multiple shoots were produced directly through shoot organogenesis from each seed explant when TDZ was added to culture medium. As many as 30 to 40 shoots were produced per seed explant in about 7 weeks from culture initiation. Different alfalfa cultivars were also tested. The established multiple shoot production protocol provides an efficient way to produce transgenic alfalfa plants, thus could significantly advance alfalfa genetic transformation.

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Guochen Yang and Marihelen Kamp-Glass

The success of genetic transformation depends on the efficiency and rehability of in vitro shoot regeneration. This research was pursued to investigate how different plant growth regulators influence alfalfa callus initiation and development, thus to establish a foundation for further development of an efficient shoot organogenesis protocol for the genetic transformation system. BA, zeatin, and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were evaluated for callus initiation and development. BA at 1 or 5 mg·liter–1, or zeatin at 5 mg·liter–1 promoted callus regeneration and further development toward shoot organogenesis. However, 2,4,5-T at 1 mg·liter–1 enhanced only callus production. These results can and will be used for further development of a shoot regeneration protocol to assist alfalfa genetic transformation.

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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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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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Guochen Yang and Marihelen Kamp-Glass

Exochorda racemosa is an ornamental shrub with white flowers that is spiraea-like, deciduous, and hardy. The buds resemble pearls. Normally it is propagated by seeds, layers, and cuttings of softwood. However, it is a slow process that takes a few years to produce a reasonable size plant for the demanding market. Our objective was to establish a successful in vitro culture and to rapidly multiply this ornamental species. Softwood explant materials were collected from a local nursery and were disinfested with 15% bleach solution and rinsed three times with sterile distilled and deionized water. In vitro cultures were established and maintained in woody plant medium (WPM) supplemented with BA at 0.1 mg·L-1, 3% sucrose, and 0.7% agar with the pH adjusted to 5.8. Then shoots were transferred to the multiplication medium containing BA, CPPU, or thidiazuron (TDZ) at various concentrations. Preliminary results show that explants cultured on medium containing TDZ produced the best shoot proliferation.

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Guochen Yang and P. E. Read

Vanhoutte's spiraea has been propagated in vitro using explants from softwood growth of dormant stems forced in a solution containing 200 mg/l 8-hydroxyquinoline citrate (8-HQC) and 2% sucrose (Yang and Read, 1989). Objectives to further utilize this system were to determine the feasibility of applying plant growth regulators (PGR) via the forcing solution to softwood growth from forced dormant stems and to study the resulting influence on in vitro culture. BA and GA3 were placed in the forcing solution at various concentrations, including a zero PGR control. Explants were cultured on Linsmaier and Skoog (LS) medium containing zero PGR or different amounts of BA or thidiazuron (TDZ) or combinations of BA and IAA. Control explants placed on LS medium supplemented with 5uM BA with or without 1 or 5uM IAA, or with 0.5 or 0.75 uM TDZ alone produced the best shoot proliferation. BA in the forcing solution stimulated micropropagation, while GA3 caused less proliferation than explants from control solutions. Forcing solutions containing PGR are useful for manipulating responses of plant tissues cultured in vitro and for studying PGR influence on woody plant physiology.

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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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Carl E. Niedziela* Jr. and Guochen Yang

Plug seedlings of lisianthus [Eustoma grandiflorum (Raf.) Shinn. `Mariachi'] and stock [Matthiola incana (L.) R.Br. `Cheerful White'] were planted on 20 Nov. 2003 followed by plug seedlings of snapdragon (Antirrhinum majus L. `Maryland') and seeds of sunflower (Helianthus annuus L. `Sunbrite') the next day. Each species was planted in four production systems (harvest lugs, lay-flat bags, pots, and polystyrene trays). Production systems were randomized in a Latin-square design with four replications of each system. Each treatment plot was 0.7 m × 1.1 m. Planting densities was 62 plants/m2 for stock and 31 plants/m2 for lisianthus, snapdragon, and sunflower. The harvest lugs were 55 cm × 37 cm × 16 cm. The lay-flat bags were 114 cm × 30 cm × 3 cm. The pots were 25 cm bulb pans. The polystyrene trays were 67 cm × 34 cm × 5 cm and contained 32 square cells. All of the containers were filled with the same tobacco germination media. The plants in the harvest lugs, lay-flat bags and pots were irrigated daily with 150 mg·kg-1 of N from 20N-4.4P-16.6K. The plants in the polystyrene trays were floated on a solution of 150 mg·kg-1 of N from 20N-4.4P-16.6K. Float solutions were monitored and adjusted weekly for volume and fertilizer concentration. Individual stems were harvested from each species at the appropriate stage of development for market. The weight and length of individual stems were recorded. Rate of growth and maturation differed between production systems and locations in the greenhouse. Detailed results will be presented.