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Ann M. Chanon and R. Daniel Lineberger

The genus Betula contains many important forest and ornamental species and a method of rapid clonal propagation of superior genotypes is needed. Thidiazuron (TDZ) is a potent synthetic plant growth regulator with cytokinin-like activity. TDZ was used to differentiate shoots after long term exposure to dichlorophenoxyacetic acid (2.4-D) as part of a larger study on clonal fidelity. Birch calli were cultured on Woody Plant Medium supplemented with 10-5 M 2,4-D for up to 30 weeks. The calli were transferred to media containing TDZ at concentrations of 10-6 to 10-9M. Most of the tissue which had not been exposed to 2.4-D differentiated shoots five weeks after being exposed to 10-6M TDZ. Increasing the of time exposure to 2.4-D or decreasing the concentration of TDZ delayed differentiation. Calli exposed to 2.4-D for more than 18 weeks rarely differentiated shoots regardless of the concentration of TDZ used.

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S. Jayasankar and U.L Yadava

Petiole discs from young leaves of female papaya (L-45) plants were cultured in MS or B5-based media containing 0, 2.25, 4.5, 11.25, and 22.5 μm 2,4-D. Compact embryogenic callus emerged from vascular tissue of petiole discs in about 3 weeks. In MS medium, 66% and 51% explants formed embryogenic callus with 11.25 and 22.5 μm 2,4-D, respectively. On the other hand, 79% explants formed embryogenic callus in B5-based medium with 4.50 μm 2,4-D. However, explants became necrotic in B5-based medium with 22.5 μm 2,4-D. Subculturing callus in auxin-free medium resulted in the development of roots or somatic embryos. Microscopic observations revealed that the roots were produced only by the callus that had retained its continuity with the vascular tissue. This investigation revealed that petioles from field grown papaya plants are potential explants for somatic embryogenesis and 2-week exposure to 2,4-D is adequate for inducing morphogenesis. Additionally, an interaction between 2,4-D and the components in the MS and B5-based media was observed.

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J.T.A. Proctor, T. Slimmon, and P.K. Saxena

Ginseng is an herbaceous perennial that grows in the understorey of deciduous hardwood forests and is also cultivated for its highly valued root. The primary method of propagation of ginseng is by seed which requires the breaking of dormancy by stratification, a process which takes 18–24 months. Investigation of factors controlling the growth and development of ginseng plants is a prerequisite to the development of a more efficient system of ginseng propagation. We have recently modulated the morphogenetic potential of geranium roots and stimulated de novo development of shoots and embryo-like structures which later formed whole plants using thidiazuron (TDZ). Our objective was to investigate the morphological changes in seedling and mature ginseng plants induced by TDZ, particularly in relation to root and shoot morphogenesis and economic yield. Applications of TDZ (0.22 and 2.20 ppm), either as foliar sprays or soil watering to greenhouse-grown seedlings over 18 weeks (2 weeks after sowing to 20 weeks when plants were harvested) induced similar effects. These responses included increased stem length and diameter, and shoot and root weight (economic yield). Single foliar applications of TDZ at 62.5 and 125 ppm to 3-year-old field-grown ginseng plants 3 months before harvest increased root biomass (economic yield) by 19% to 23%. Roots of TDZ-treated seedlings and 3-year-old field-grown plants developed thickened secondary roots on the upper part of the taproot. The root-like structure of these secondary roots was confirmed by histology. In addition, TDZ treatments induced adventitious buds on the shoulder of 3-year-old roots. These buds developed into shoots to give multi-stem plants following a period of dormancy, which was overcome with GA3 (gibberellic acid) treatment before planting.

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Michael J. Tanabe and Nicole Wakida

Noni, Morinda citrifolia, is receiving a lot of attention for its potential medicinal effects. Hawaii is an ideal growing environment for this plant, where it has been used for many purposes, including medicinal ones, by ancient Polynesians. Currently, there is a rapidly developing noni industry in the state of Hawaii. Propagation of this plant is almost exclusively by seeds, and germination generally requires a couple of months without preconditioning or about a month if mechanically scarified. We developed an in vitro protocol that significantly improves percent germination rate by altering incubation temperature and the in vitro culture basal medium. Germination time was decreased to 4 days when the embryo was extracted and exposed to 31 °C. A basal medium containing 1/2 Murashige and Skoog (M&S) salts was the most effective in reducing germination time and increasing percent germination. Stem pieces obtained from in vitro-propagated seedlings produced callus when explanted in 1/2 M&S containing various levels of naphthalene acetic acid (NAA). The most effective treatment was 0.5 μm NAA and the least effective treatment was 2 μm NAA. Treatments without NAA did not produce callus. Calli treated with 4.40 μm 6-benzylaminopurine (BA) or 8.80 μm BA were the most effective in promoting caulogenesis. We also demonstrated that the number of first generation seedlings produced from each embryo could be increased by treatment with 8.80 μm BA.

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Mohamed F. Mohamed, Dermot P. Coyne, and Paul E. Read

Plant regeneration has been achieved in two common bean lines from pedicel-derived callus that was separated from the explant and maintained through successive subcultures. Callus was induced either on B5 or MS medium containing 2% sucrose and enriched with 0.5 or 1.0 mg thidiaznron/liter alone or plus various concentrations of indoleacetic acid. The presence of 0.07 or 0.14 g ascorbic acid/liter in the maintenance media prolonged the maintenance time. Up to 40 shoot primordia were observed in 4-week-old cultures obtained from 40 to 50 mg callus tissues on shoot-induction medium containing 1-mg benzyladenine/liter. These shoot primordia developed two to five excisable shoots (>0.5 cm) on medium with 0.1-mg BA/liter. A histological study confirmed the organogenic nature of regeneration from the callus tissues. The R2 line from a selected variant plant showed stable expression of increased plant height and earlier maturity. Chemical names used: ascorbic acid, N- (phenylmethyl)-1H-pnrin-6-amine [benzyl-adenine, BA], 1H-indole-3-acetic acid (IAA), N- phenyl-N'-1,2,3-thiadiazol-5-ylurea [thidiazuron, TDZ].

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Guirong Li, Ran Quan, Chaohui Yan, Xiaojin Hou, and Huiling Hu

Grape (Vitis vinifera) is among the world’s most important fruit crops and is a commonly used woody plant for genomics and post-genomics research. NAC transcription factors play central roles in plant growth and development, floral organ morphogenesis, and responses to biological stress. It is therefore important to identify key transcription factors from grape and clarify their mechanisms of action to generate genetic resources for grape molecular improvement. Our research group previously cloned a NAC transcription factor from V. vinifera ‘Yatomi Rosa’ [drought and leaf roll gene 1 (DRL1)] and demonstrated that it caused dwarfing of tobacco (Nicotiana benthamiana) plants when overexpressed. In the present work, we demonstrate that overexpression of DRL1 in transgenic tobacco delays flowering time and markedly reduces pollen viability. Furthermore, crosses between male DRL1 transgenic tobacco and female wild-type tobacco exhibit substantially lower fruit set, fruit and seed weights, fruit and seed shape indices, and seed germination rates than selfed wild-type plants or crosses with a transgenic female parent. DLR1 overexpression strongly influences flowering time and reproduction in transgenic tobacco, primarily through its effects on pollen development. These results provide a foundation for further functional characterization of DLR1 in grape.

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Michael E. Compton and D.J. Gray

Cotyledon explants of four watermelon [Citrullus lanatus (Thunb.) Mataum. & Nakai] breeding lines (F92U8, SP90-1, SP90-2, and SP90-4) were prepared from mature seed or from 2-, 4-, 6-, 8-, or 10-day-old seedlings. Explants were incubated on shoot regeneration medium for 8 weeks followed by 4 weeks on shoot elongation medium. The four genotypes differed in their ability to produce shoots at each explant age. The highest frequency with which F92U8 (66%) and SP90-2 (60%) explants produced shoots was for 2-day-old seedlings. Fewer explants formed shoots when established from mature seed or seedlings older than 2 days. In contrast, the percentage of SP90-4 explants that produced shoots was highest when cotyledons were obtained from 4-day-old seedlings (40%), but the response was less than the optimum for F92U8 and SP90-2. SP90-1 cotyledon explants exhibited the poorest response of the four breeding lines (<11% produced shoots), with little difference in response among the explant ages tested. The number of shoots per responding explant also depended on the age of the explant source. Explants from 2- to 4-day-old seedlings produced the most shoots. Fewer shoots formed on cotyledons from mature seed or seedlings older than 4 days.

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James A. Kapaun and Zong-Ming Cheng

Plants were regenerated from leaf tissue of greenhouse-grown seedlings of Siberian elm (Ulmus pumila L.). Shoot regeneration was induced on Murashige and Skoog (MS) medium containing 5 to 10 μm of BA. Up to 55% of the leaf explants formed shoots with an average of 2.4 shoots per explant. Addition of 2.5 or 5 μm of IBA failed to enhance regeneration. Thidiazuron at 0.5 or 1.0 μm also induced shoot regeneration, but the shoots failed to elongate as well as shoots regenerated from media containing BA. Incubation in darkness for 7, 14, or 21 d had little effect in promoting shoot regeneration, except that incubation for 21 d increased shoot regeneration on the medium with 5.0 μm BA. Genotypes differed in shoot regeneration potential, with regeneration frequencies ranging from 13% to 55%. Regenerated shoots were micropropagated on Driver and Kuniyuki Walnut medium. Ninety percent of microcuttings rooted directly in potting soil. This regeneration system will be valuable for genetic transformation and cell selection of Siberian elm. Chemical names used: 6-benzylaminopurine (BA); indole-3-butyric acid (IBA); N-phenyl-N′ -1,2,3-thiadiazol-5-ylurea (thidiazuron, TDZ).

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Robert L. Geneve, Wesley P. Hackett, and Bert T. Swanson

Exogenous ethylene could not substitute for NAA to induce adventitious root initiation in juvenile petiole explants of English ivy (Hedera helix L.), indicating that the action of auxin-stimulated root initiation was not directly mediated through ethylene production. Mature petioles did not initiate roots under any auxin or ethylene treatment combination. Ethephon or ACC supplied at 50 or 100 μm was inhibitory to NAA-induced root initiation in juvenile petioles. The pattern of ethylene production stimulated by NAA application was significantly different in juvenile and mature petioles. Ethylene evolution by juvenile petioles declined to near control levels during from 6 to 12 days after NAA application. Reduction in ethylene production was due to reduced availability of ACC in juvenile petioles. Mature petioles continued to produce ethylene at elevated levels throughout the course of the experiment. Ethylene does not appear to play a significant role in the differential root initiation response of juvenile and mature petioles treated with NAA. However, ethylene appeared to have an inhibitory effect during root elongation stages of adventitious root development in juvenile petioles. Chemical names used: 1-aminocyclopropane-1-carboxylic acid (ACC); 1-napthaleneacetic acid (NAA); 2-chloroethylphosphonic acid (ethephon).

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Dennis P. Stimart and John C. Mather

Cotyledons from developing 6- to 8-week-old embryos of Liatris spicata (L.) Willd. (blazing star) were cultured on Murashige and Skoog medium containing 0, 0.4, 4.4, or 44.4 μm BA or 0, 0.2, 2.2, or 22.2 μm TDZ to induce adventitious shoot formation. The highest percentage of cotyledons forming the most shoots was on medium containing 2.2 μm TDZ. Cotyledon-derived callus cultured on medium containing 4.4 μm BA formed ≈16 times more adventitious shoots than on 2.2 μm TDZ. Adventitious shoots derived from cotyledons or callus produced roots when placed on MS medium containing 5.0 μm IBA. Regenerated plants that flowered in the field appeared homogeneous. Chemical names used: N6-benzyladenine (BA), thidiazuron (TDZ), indole-3-butyric acid (IBA).