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Samir C. Debnath

The growth and development of lingonberry (Vaccinium vitis-idaea L.) plants propagated either by conventional softwood cuttings or by in vitro shoot proliferation from nodal explants and by shoot regeneration from excised leaves of micropropagated shoots, were studied in cultivars `Regal', `Splendor', and `Erntedank'. Significant differences were observed between the treatments. After 3 years of growth, the in vitro-derived plants produced more stems, leaves, and rhizomes than the conventional cuttings which rarely produced rhizomes. In vitro culture on nutrient medium apparently induces the juvenile branching characteristics that favor rhizome production. This increase in vegetative growth and rhizome yield of in vitro-derived plants over stem cuttings varied among genotypes.

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Chunsheng Lu, Yiqin Ruan, and Mark Bridgen

Micropropagation has been used to rescue Leontochir ovallei, an endangered Chilean species in the Alstroemericeae. Cultures were initiated by aseptically germinating seeds of Leontochir on a medium containing 1/10 MS salts and vitamins and 0.3% sucrose. Three types of cytokinins (BAP, 2-iP and kinetin) at four concentrations (0, 2, 4, and 8 uM) were studied for shoot proliferation. In the 4 uM BAP treatment, new shoots were produced at an average of six per culture after four weeks of culture. Overall, there was an average of four shoots/culture/4 weeks for all BAP treatments. This was significantly higher than the 2-iP and kinetin treatments. Moreover, the increase of culture fresh weight over time was significantly greater in BAP treatments than those in other treatments. A rooting study compared the effect of NAA and IBA on root initiation. Over 85% of the cultures in 10 and 20 uM NAA treatments produced healthy and large roots. This was significantly higher than the 10 and 20 uM IBA treatments. In summary, a concentration of 4 uM BAP combined with 1 uM IBA in MS salts and vitamins supplemented with 146 mg glutamine/l is the best for shoot proliferation of leontochir; an MS basal medium containing 10 uM NAA is the best for root initiation. Micropropagated plantlets have been successfully transplanted into the greenhouse for further genetic and breeding studies.

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Maritza I. Tapia and Paul E Read

It has been previously demonstrated that thidiazuron (TDZ) enhanced the regeneration and multiple shoot proliferation of vinifera grape cultivars. To determine the effect of TDZ on the multiplication of hybrid grapes, in vitro nodal segments from cultivars Chancellor, Leon Millot, and Valiant were cultured on MS medium supplemented with 0, 0.01, 0.05, 0.1, 0.5, and 1.0 mg TDZ/liter. After 1 month, the higher percentage of rooted shoots was obtained from the explants cultured in medium containing the lowest concentration of TDZ (0.01 mgliter–1) independent of the genotype. Multiple shoot proliferation was favored by high concentrations of TDZ (0.5 and 1.0 mgliter–1). An average of 0.39 and 0.39 shoots, respectively, was obtained from `Chancellor' cultures, 0.56 and 0.59 from `Leon Millot', and 1.93 and 2.38 from `Valiant'. Vitrification and teratological structures were observed in all the cultures of the three genotypes, but less vitrification occurred in `Valiant' plantlets.

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Thomas W. Zimmerman and Ralph Scorza

This study examined stage I peach shoot growth under various photoperiods in combination with different vessel closures and compared the influence of BA and Thidiazuron (TDZ) on peach shoot growth during stage II. The basal salts were as described by Almehdi and Parfitt (1986) with 1.0 μM BA, 0.02 μM IBA, 2% sucrose, 0.1% gelrite and 0.4% agar. Shoot growth of peach clone B612615, as determined by leaf number after one month, was similar in vessels capped with Kim-Kaps, Kaputs or PM caps. Plastic foam Identi-Plugs resulted in desiccation of the medium and stressed shoots with reduced growth. A 4 h light/2 h dark photoperiod four times a day provided better growth during stage I than a 16 h light/ 8 h dark photoperiod. For stage II, established shoots of Suncrest, Georgia Bell and Evergreen were grown on MS medium supplemented with 0.02 μM IBA in combination with 1.0 or 10 μM BA or 0.1, 1.0 or 10 μM TDZ. TDZ produced excessive callus resulting in minimal shoot proliferation. Shoot proliferation from axillary buds was greatest with 10 μM BA.

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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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Sudarsono and Ronald G. Goldy

Four muscadine grape (Vitis rotundifolia Michx.) cultivars (Carlos, Noble, Regale, and Tarheel) were evaluated for their ability to be cultured in vitro. Axillary buds were placed on Murashige and Skoog medium as modified by Chee. Different levels of benzylaminopurine [(BA) 0.5 to 10.0 μm], kinetin [(KIN) 0.5 to 5.0 μm], and thidiazuron [(TDZ) 0.5 to 11.3 μm], and different explant positions were evaluated for their effect on in vitro explant establishment and shoot production. Thidiazuron (2.3 to 4.5 μm) alone or in combination with BA (1.0 to 5.0 μm) or KIN (1.0 or 5.0 μm) was effective for establishing axillary buds. Similar levels were also effective for promoting shoot proliferation. Explants originating from the 10 basal nodes of a shoot with at least 25 nodes gave better shoot proliferation than explants originating from the 10 distal nodes. Chemical names used: 6-benzylaminopurine, 6-furfurylaminopu. rine (kinetin):N -phenyl-N'-l,2,3 -thiadiazol-5-y lurea (thidiazuron).

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Michael E. Kane, Edward F. Gilman, Matthew A. Jenks, and Thomas J. Sheehan

Procedures for in vitro establishment, rapid shoot proliferation, and ex vitro plantlet acclimatization of Cryptocoryne lucens de Witt were determined. Shoot cultures were established from surface-sterilized shoot tips cultured on Linsmaier and Skoog salts and vitamins medium (LS) solidified with 0.8% (w/v) agar and supplemented with 2.0 μm BA and 0.5 μm NAA. The effect of BA (0 to 20 μm) and 0.5 μm NAA on shoot multiplication from single-node and clustered triple-node shoot explants was determined after 35 days. The most efficient shoot proliferation (7.7 shoots/explant) occurred from single-node shoot explants cultured on LS + 20 μm BA and 0.5 μm NAA. Maximum plantlet establishment was achieved by direct sticking of triple-node (cluster) microcuttings in either soilless planting medium or polyurethane foam cubes. Production of highly branched salable plants from microcuttings was possible within 18 weeks. Chemical names used: N-(phenylmethyl) -1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA).

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Francisco Molinar Jr., Wayne A. Mackay, Marisa M. Wall, and Manuel Cardenas

Experiments were conducted to develop a clonal propagation system for agarita (Berberis trifoliata Moric.). Actively growing agarita shoots were collected from a mature plant at the Texas A&M Univ. Research and Extension Center in El Paso and successfully established on a basal medium consisting of woody plant medium (WPM) salts and Murashige and Skoog vitamins, sucrose at 30 g·L–1, and 0.8% Phytagar supplemented with 11.1 μm BA. Cytokinins (benzyladenine, kinetin, and thidiazuron), subculture period, and age of cultures were tested. The optimal shoot proliferation conditions were WPM basal medium supplemented with 5.5 μm BA and a subculture period of 4 weeks. Culture age did not affect shoot proliferation but did affect rooting. Preliminary experiments with 1.0 μm NAA resulted in nearly 100% rooting of microshoots <6 months old. Shoots from 21-month-old cultures had to be placed on a cytokinin-free medium before successful rooting. On basal medium supplemented with NAA (5.4 μm), 68% of the microshoots rooted with an average of 1.2 secondary roots per microshoot. Chemical names used: N-(phenylmethyl)-1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA); N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ); 6-furfurlaminopurine (kinetin).

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

Clonal micropropagation studies with silver maple (Acer saccharinum L.) included experiments with various shoot. explant types, cytokinins, and stock plant maturation levels. These trials led to successful explant establishment, axillary shoot proliferation, rooting of microshoots, and establishment of plantlets in the greenhouse. Overall, the best cytokinin tested was the phenylurea derivative TDZ. Shoot proliferation on juvenile explants was poor with kinetin, 2iP, and BA. Only zeatin at 10 μm was comparable to TDZ. TDZ at 10 nm was optimal for both juvenile and adult nodal explants. Juvenile explants that were held in vitro for 4 months commonly had at least 60 axillary shoots that could be subculture or excised for rooting. Microshoots rooted within 2 weeks. Following rooting, silver maple plantlets could be transplanted into a growing medium and placed directly onto a greenhouse bench. Studies were also conducted on rooting stem cuttings (macropropagation). Single nodes from juvenile plants rooted under intermittent mist, regardless of auxin application; however, shoot-tip cuttings from adult trees rooted best when auxin in ethanol solution was applied. Chemical names used: N- phenyl- N' -1,2,3 -thiadiazol-5-ylurea (thidiazuron, TDZ), N- (2-furanylmethyl)-1H-purin-6-amine (kinetin), isopentenyladenine (2iP), benzyladenine (BA), (E)-2-methyl-4-(1H-purin-6-ylamino)-2-buten-1-ol (zeatin).

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Guochen Yang, Marihelen Kamp-Glass, and Paul E. Read

American chestnut (Castanea dentata) is one of the United States' most valuable resources for its nuts and timber. Many scientists are exploring genetic transformation techniques to improve chestnut blight resistance in addition to conventional breeding. In vitro shoot production must be first obtained and optimized in order to establish an efficient transformation system. Although shoot proliferation has been achieved, chestnut is still considered difficult for tissue culture with poor rooting. Therefore, this research has focused on improving rooting ability of micropropagated chestnut shoots. In vitro shoot production was established and maintained in WPM supplemented with 0.1 mg/l BA, 3% sucrose, and 0.7% agar with the pH adjusted to 5.8. The shoots were then transferred to rooting medium containing the same components as for shoot proliferation plus an auxin at various concentrations. Right after placing shoots onto rooting medium, a very thin layer (5 ml) of the same auxin (diluted) was added to provide a quick stimulation of rooting. Detailed discussion will be presented.