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Boris Andrés Bran Barrientos and Jong-Yi Fang

depth the influence of culture media and incubation conditions on the speed of seed germination and seedling development. For commercial and amateur growers, as well as future breeders of S. plicata , a tissue-culture protocol that ensures rapid

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Youping Sun, Donglin Zhang, and John Smagula

Nodal segments containing one axillary bud (1 to 1.5 cm) were disinfected using 10% bleach and were established on a Murashige and Skoog (MS) medium without hormones at 27 °C and with a 16-h photoperiod. The sprouted shoots (≈1.0 cm) were cultured on a MS medium supplemented with 6-benzylaminopurine (BAP), kinetin (KIN), or zeatin (ZT) at 2.3, 4.5, 9.1, or 18.2 μM. After 38 d, ZT and BAP significantly induced multiple shoot formation with multiplication rates of 4 to 6, whereas the multiplication rate of KIN was less than 2. Shoots cultured on ZT grew significantly taller than those on BAP and KIN. The height of the longest shoots treated with ZT was 4.6 cm, which was 1.6 to 2.2 times greater than those treated with BAP or KIN. To induce rooting, shoots (≈2 cm) were subcultured on one-fourth strength MS (1/4 MS) medium containing either 3-indolebutyric acid (IBA) or 1-naphthylacetic acid (NAA) at 2.6, 5.1, or 10.3 μM. Adventitious roots formed in vitro after 2 to 4 weeks. IBA at 10.3 μM produced the best rooting (100%) compared with other treatments after 38 d of culture. The average number of roots per shoot for IBA was ≈15, which was 1.6 to 3.1 times as many as that of other treatments. All rooted plantlets were then transplanted into a mix of peatmoss and perlite (1:1 v/v) and acclimatized in a mist system. Average plantlet survival was 73.6% after 35 d. After acclimatization, they were grown in a pot with Metro-mix under greenhouse conditions for 10 weeks where 95% of plants survived and grew up to 6.8 cm high. The micropropagation procedure, i.e., nodal segments containing one axillary bud proliferated on MS with 4.5 μM ZT followed by in vitro rooting on 1/4 MS plus 10.3 μM IBA, could be used for commercial mass production of new inkberry cultivars.

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Joe-Ann McCoy and N.D. Camper

Hypericum perforatum L. (St. John's Wort) has an extensive history as an important medicinal herb used for the treatment of neurological and depressive disorders (Linde et al., 1996). The objective of this study was to establish an in vitro tissue culture protocol for St. John's Wort. Nodal segments, axillary buds, and leaf disc explants produced multiple shoots and callus on Murashige and Skoog minimal organics medium supplemented with combinations of indoleacetic acid (IAA; 0.57, 2.85, 5.71 μm) and benzylaminopurine (BA; 2.22, 4.44, 8.88 μm). Shoot production occurred on all combinations of IAA/BA tested and was significantly less in treatments without hormones. Callus production was higher on treatments containing 2.85 μm IAA + 4.44 μm BA, or 5.71 μm IAA + 8.88 μm BA. Shoots transferred to hormone-free medium at 8 weeks formed roots by 12 weeks. A micropropagation protocol was established for St. John's Wort using mature plants as the explant source.

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Antonio Figueira, Anna Whipkey, and Jules Janick

Axillary shoots of cacao (Theobroma cacao L.), induced in vitro with cytokinins (BA or TDZ), elongated and produced leaves only in the presence of cotyledons and/or roots. Detached axillary shoots, which do not grow in `vitro under conventional tissue culture protocols, rooted with auxin and developed normally in vivo. Detached axillary shoots from cotyledonary nodes and single-node cuttings from mature plants were induced to elongate and produce normal leaves in the presence of 20,000 ppm CO2 and a photosynthetic photon flux density (PPFD) of 150 to 200 μmol·s-1·m-2. Subculture nodal cuttings continued to elongate and produce leaves under elevated CO2 and light levels, and some formed roots. Subculture of microcuttings under CO2 enrichment could be the basis for a rapid system of micropropagation for cacao. Chemical names used: N -(phenylmethyl) -1 H -purin-6-amine (BA); 1 H -indole-3-butyric `acid (IBA); α -naphthaleneacetic acid (NAA); thidiazuron (TDZ).

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Justin A. Schulze, Jason D. Lattier, and Ryan N. Contreras

A tissue culture protocol was developed to germinate immature Prunus lusitanica seeds in vitro. The study was conducted by first identifying the best media for germination, followed by investigating effects of seed conditioning. In Expt. I, seeds were collected 12 weeks after pollination (WAP) ± 1 week and placed on media after removing the pericarp. Eight different MS media (Murashige and Skoog, 1962) were tested (M1–M8) containing two concentrations each of 6-benzylaminopurine (BA), gibberellic acid (GA3), and sucrose. The longest shoots resulted from M4 (1.45 µm GA3, 6 µm BA, and 30 g·L−1 sucrose), followed by M1 (0 µm GA3, 3 µm BA, and 30 g·L−1 sucrose). Radicle and shoot emergence was greater than or equal to 90% for M1, M3, and M4 after a stratification treatment. In Expt. II, M1 was used to test root and shoot emergence at 6, 9, and 12 WAP, with and without cold stratification. Little success was seen 6 and 9 WAP, with only callus development in 6 WAP, nonstratified seed. Cold stratification increased shoot emergence in the 12 WAP group from 4% to 28%, appearing to be critical for shoot emergence. If the cotyledons are retained on the seed, future efforts to expedite breeding of P. lusitanica using in vitro germination should not be collected before 12 WAP and will benefit from cold stratification before germinating on M1 or M4. Chemical names: 6-benzylaminopurine (BA), gibberellic acid (GA3).

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A. Raymond Miller, Joseph C. Scheereus, Patricia S. Erb, and Craig K. Chandler

A tissue culture protocol was developed that increased the germination percentage and decreased the lag time to germination for strawberry (Fragaria x ananassa Duch.) achenes. This technique involved cutting surface-sterilized achenes across the embryo axis then placing the shoot apex/radicle-containing sections on semisolid Murashige and Skoog medium lacking hormones. Cut achenes began germinating 5 days after culture and achieved maximum germination (97% to 100%) in less than 2 weeks, compared to whole achenes, which began to germinate 7 to 10 days after sowing and required more than 7 weeks for maximum germination (<50%). Enhanced germination of cut achenes was a general phenomenon since achenes from 231 hybrid crosses responded similarly. Following placement on culture medium, cut achenes could be stored up to 8 weeks at 4C then removed to 27C, where germination and seedling development occurred at percentages and rates comparable to freshly cut achenes. Achenes did not require stratification before cutting to exhibit increased germination. Nearly 100% of the achenes from freshly harvested red-ripe, pink and white strawberries germinated after cutting and culture, although cut achenes from white and pink berries germinated more slowly than those from red-ripe berries. Achenes from green berries, whether whole or cut, did not germinate. This method of “embryo rescue” could be used to generate more seedlings from poorly germinating hybrid crosses, would considerably decrease the time from sowing to seedling production compared to traditional means, and would produce seedlings of uniform age for subsequent field evaluation.

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Seong Min Woo and Hazel Y. Wetzstein

Georgia plume (Elliottia racemosa Muhlenb. ex. Elliott) is a rare deciduous shrub or small tree. It has sustained severe loss of habitat and its range is now restricted to a limited number of sites in the state of Georgia. Tissue culture protocols have been developed as a means to propagate and conserve this threatened species using leaf explants induced on medium supplemented with 10 μm thidiazuron (TDZ) and 5 μm indole-3-acetic acid (IAA). Bud-like clusters, elongated embryo-like protrusions, and shoot-like structures were produced from the leaf explants. Morphological and histological evaluations of cultures during induction and development were conducted using light microscopy of sectioned material and scanning electron micrography. Histology of explant tissues indicates that plant regeneration of Georgia plume occurs through a shoot organogenesis pathway that involves the formation of actively dividing meristematic regions originating in subepidermal cell layers that proliferate to form protuberances on the explant surface. Numerous well-formed shoot apical meristems with leaf primordia are produced, as well as fused shoot-like structures. Elongated, embryo-like structures had various degrees of shoot apex development. Evaluations of serial sections found that they lacked a defined root apex, and that basal portions were composed of parenchymatous files of cells with a broad point of attachment to the parent tissue. The lack of bipolarity and a root pole signifies that true somatic embryogenesis does not occur.

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James Robert Ault and Sandy S. Siqueira

. Some of the research tools being used in this germplasm improvement program include embryo/ovule rescue from wide interspecific crosses, polyploid induction, and in vitro propagation. These studies depend on effective tissue culture protocols. There

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container production of these woody shrubs in Kentucky. Tissue Culture Protocols for Conserving Oak Species Oaks are economically and ecologically valuable around the world, but many species are under threat. Oak secies cannot be preserved through

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Miles Schwartz Sax, Nina Bassuk, and Mark Bridgen

stool-bed rooting method. To overcome this limitation, tissue culture protocols were trialed to determine if in vitro methods could successfully be used to clonally propagate UHI hybrid oaks. The use of oak tissue culture methods used to grow plants