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Linda J. Walker, R.B. Rogers, and M.A.L. Smith

In vitro cell cultures of huckleberry and bilberry are sources of phytochemicals for use as food colorants and bioactive chemopreventives. Shoot cultures provide a convenient, presterile source of explants for production of callus rich in extractable pigments or other chemicals. Efficient callus formation only occurs with good-quality shoots. In this study, liquid and gelled support systems were compared in terms of their effect on shoot growth. Gellan gum-based support resulted in excellent shoot proliferation and suitable shoot length for huckleberry cultures, whereas bilberry performed slightly better on agar and agar/gellan gum support. Bilberry had a more-rapid growth rate than huckleberry. Hyperhydricity was found with the use of rafts for both species. These shoot cultures have been used as vegetative explants for callus, and have produced vivid anthocyanins in solution cultures.

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John E. Preece, Carl A. Huetteman, W. C. Ashby, and Paul L. Roth

During the research phase, a system was developed to clonally micropropagate silver maple. Explant performance was best on DKW medium with 10 nM thidiazuron, and explants commonly developed 1 7 shoots after three months and over 60 shoots that could be rooted after four months in vitro. Plants were rooted (>90%) and acclimatized under intermittent mist and transplanted to an outdoor nursery bed. However, results were different during the production phase when 90 clones were propagated. Shoot proliferation rates were lower, differences in clonal response and worker efficiency were apparent, mass rooting under mist was inconsistent and acclimatization problems arose. The mean rooting was 46% under mist because of uneven coverage. Only 56% of rooted plantlets acclimatized which resulted in an overall efficiency of 26%. Partial solutions included root initiation in vitro, and use of fog for acclimatization.

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Richard L. Bell

Preconditioning effects of cytokinin in the shoot proliferation medium on explant quality and subsequent adventitious regeneration of `Bartlett' and `Beurre Bosc' pear were investigated. The basal medium for regeneration consisted of half-strength MS macro- and micronutrients, MS organics, 30 g·liter–1 sucrose, 6 g·liter–1 agar, and 10 μM thidiazuron (TDZ), and 1 μM NAA. Leaves from BAP medium were more effective than those from media with 2-iP or kinetin in spite of the increased leaf size of shoots cultured with 2-iP (28% vs. 10%). Use of leaves from in vitro-rooted shoots did not increase regeneration frequency (19.5% vs. 31%) of `Bartlett'. Actively expanding leaves are more suitable explants than larger, fully expanded leaves. Liquid medium overlays and incubation in liquid medium decreased regeneration frequency when compared with agar-solidified medium. Among auxins in regeneration induction phase media, IBA (0.5 or 1.0 μM) resulted in greater regeneration than NAA.

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Steve McCulloch

Briggs Nurseries, Inc. has used micropropagation as method of vegetative propagation for over 20 years. Genetic stability and uniformity of plants that are produced and sold is of the utmost concern to the commercial plant propagator. Genetic stability may be accomplished by ensuring that all shoots formed in vitro are of axillary origin and by reducing shoot proliferation rates through the use of lower cytokinin concentrations in the culture medium. Excision and removal of callus during transfer is also necessary to ensure that shoots develop from axillary buds. Various factors that may influence genetic variability and its frequency of in vitro derived plants will be discussed with an emphasis on how to reduce them. Three sources of variation with tissue culture derived plants will also be reviewed (Swartz, 1991): a) source plant variability, b) genetic changes in vitro, and c) epigenetic or physiological adaptation.

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Line Brissette, Laurence Tremblay, and Daniel Lord

Bud cultures from nonjuvenile field clones of lowbush blueberry (Vaccinium angustifolium Ait.) were established on Z-2 medium with 59 μm 2iP. Reversion to juvenile characteristics with small and rounder leaves occurred only on two explants after 19 weeks in culture. These shoots grew vigorously and could be easily subcultured. The number of shoots of one clone doubled every 23.3 days. Reducing the 2iP concentration to 12.3 and 24.6 μm reduced shoot proliferation, but permitted better shoot elongation. After 5 weeks in a mix of 3 peat: 2 vermiculite: 1 perlite, shoots >20 mm rooted better than shoots measuring 10 to 20 mm. Chemical names used: N-(3-methyl-2-butenyl)-1-H-purine-6-amine (2iP); 1H-indole-3-acetic acid (IAA).

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J.J. Le Roux and J. Van Staden

Two cold-tolerant species (Eucalyptus macarthurii Deane et Maiden and E. smithii R.T. Baker), a cold-tolerant hybrid (E. macarthurii×E. grandis Hill ex Maiden), and E. saligna Sm. were propagated in vitro from nodal explants collected from field-grown seedlings and from clonal hedges. Shoot growth was initiated on modified Murashige and Skoog (MS) medium containing BA at 0.1 mg·liter-1. Modified MS medium with BA (0.2 mg·liter-1) and NAA (0.01 mg·liter-1) was most effective in promoting shoot proliferation. Root initiation was achieved on half-strength modified MS medium with 2 mg IBA/liter. Rooted plants were hardened and established in the field. Chemical names used: N-(phenylmethyl)-1EZ-purin-6-amine (BA); 2-(1-naphthyl)acetic acid (NAA); 1H-indole-3-butyric acid (IBA).

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James R. Ault

Shoot formed in vitro from twin-scale explants of Eucomis autumnalis (Mill.) Chitt., E. comosa (Houtt.) Wehrh., and E. zambesiaca Bak. cultured on Murashige and Skoog (MS) basal medium containing 0.0, 4.4, 11.1, or 22.2 μm BA and 0.0 or 5.4 μm NAA. In all three species, shoot proliferation was obtained from single-shoot explants subcultured on medium supplemented with 4.4, 11.1, or 22.2 μm BA and 0.0 or 5.4 μm NAA. Shoots of all three species rooted readily on MS medium supplemented with 0.0, 2.7, 5.4, or 10.8 mm NAA. Overall rooting percentages were 95%, 98%, and 100% for E. autumnalis, E. comosa, and E. zambesiaca, respectively. Plant survival for rooted shoots of all three species was 100% following transfer to a 1 perlite: 1 peat (v/v) medium in the greenhouse. Chemical names used: 6-benzyladenine (BA); 1-naphthaleneacetic acid (NAA).

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Karen L. Thies and Clinton H. Graves Jr.

A meristem micropropagation system was developed to produce Agrobacterium -free muscadine grape. Meristems were cultured on a modified Woody Plant Medium (mWPM) supplemented with 0.45 μm BAP. After 2 weeks, cultures were transferred to mWPM containing 8.92 μm BAP to enhance shoot proliferation. Propagules were subsequently subdivided and transferred to fresh medium at 2- to 4-week intervals. New shoots were excised and inserted in mWPM supplemented with 0.57 μm IAA to promote root formation. This method has been successfully used to produce Agrobacterium -free plants of muscadine cultivars Carlos, Doreen, Jumbo, Magnolia, and Sterling for research purposes and for a foundation planting in Mississippi. Chemical names used: benzylaminopurine (BAP); indole3-acetic acid (IAA).

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Philip W. Clayton, John F. Hubstenberger, Gregory C. Phillips, and S. Ann Butler-Nance

Micropropagation of 11 rare or endangered cacti species belonging to the subtribe Cactinae was achieved by rooting of proliferated axillary shoots. Shoot tip explants were obtained from seedlings of Escobaria missouriensis D.R. Hunt, E. robbinsorum (Earle) D.R. Hunt, Sclerocactus spinosior (Engelm.) Woodruff & L. Benson, and Toumeya papyracantha (Engelm.) Br. & Rose, and from mature plants of Mammillaria wrightii Engelm., Pediocactus bradyi L. Benson, P. despainii Welsh & Goodrich, P. knowltonii L. Benson, P. paradinei B.W. Benson, P. winkleri Heil, and S. mesae-verdae (Boissevain) L. Benson. Three or four species were used in each of a series of experiments investigating the effects of basal media and auxin and cytokinin types and concentrations on axillary shoot proliferation. Low or no auxin but moderate to high cytokinin concentrations were required for axillary shoot production. All species rooted spontaneously on hormone-free media; however, several species rooted better on media containing auxin. All species were re-established in the greenhouse.

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James R. Ault

Shoot tip and stem segment explants collected from greenhouse-maintained plants of Hymenoxys acaulis var. glabra were cultured in vitro for shoot initiation on a Murashige and Skoog (MS) medium supplemented with 30 g·L-1 sucrose, 2.5 μm BA, and 7 g·L-1 agar at a pH of 5.7. Unbranched shoot explants were subcultured to MS medium with 0.0, 0.5, 1, 2, 4 or 8 μm BA for shoot proliferation. A maximum of 10.3 shoots per explant was produced on the medium with 2.0 μm BA. Nonrooted shoots were subcultured to MS medium with 0.0, 0.5, 2, or 8 μm K-IBA for rooting. Maximum rooting was 90% on MS medium with 0.5 μm K-IBA. Rooted shoots were greenhouse-acclimatized for 10 days. Overall survival was 75%. Chemical names used: 6-benzyl adenine (BA); potassium salt of indole-3-butyric acid (K-IBA).