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Hany M. El Naggar, Paul E. Read, and Susan L. Cuppett

Rosemary (Rosmarinus officinalis) belongs to the Lamiaceae family, and is native to the Mediterranean and one of the most important medicinal herbs containing antioxidants in its leaves. One of the most important antioxidants is rosmarinic acid (RA). The aim of this study was to test the concentration of (RA) and chlorophyll content in leaves and callus of five successive subcultures of five different genotypes of rosemary. They were: 1) `Majorca'; 2) Rosmarinusofficinalis; 3) `Pine Scented'; 4) `Madeline Hill', and 5) APR. It was found that the highest concentration of RA in leaves was in `Pine Scented', while the lowest concentration was for APR and `Madeline Hill'. However, in the callus the highest RA concentration was for Rosmarinusofficinalis in the second subculture and `Madeline Hill' in the third subculture, while the lowest RA concentration was for `Majorca', `Pine Scented', and APR. The RA concentration in callus declined after the second and the third subculture for Rosmarinusofficinalis and `Madeline Hill', respectively. We concluded that it is preferred to use `Pine Scented' for RA extraction from the leaves while for RA extraction from callus it is better to use Rosmarinusofficinalis in the second subculture or `Madeline Hill' in the third subculture.

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Daksha Sankhla, Tim D. Davis, and N. Sankhla

`German Red' is a thermotolerant cultivar of carnation (Dianthus caryophyllus) that blooms almost year-round in Texas. This study was initiated to evaluate the feasibility of inducing somatic embryos for use in gene transfer Studies and rapid mass propagation. Internodal explants, obtained from microshoots of plantlets cultured on MS medium containing 5 μM benzyladenine (BA) and 0.5 μM naphthaleneacetic acid (NAA), were used to initiate callus. Callus formation was induced on MS medium containing 3% sucrose, 0.1% casein hydrolysate and 2,4-D (1-5 μM) alone or in combination with BA (2 or 4 μM) or kinetin (2 or 4 μM). After about 5 weeks, the callus was transferred to either semisolid or liquid MS basal medium with or without kinetin and BA. Within 20-30 days, pro-embryogenic callus masses were observed. The embryos developed from white embryonic tissue and exhibited typical stages of embryogenesis. After 5 weeks, up to 70% of the cultures grown in the liquid medium with or without BA exhibited a profusion of embryo-like structures. Because only a small percentage of these developed into plantlets, more work is needed to enhance conversion frequency.

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Jameel M. Al-Khayri, Teddy E. Morelock, and Edwin J. Anderson

Cowpea, or southernpea, is an important food legume that provides a source of high-quality protein, especially in the mature seeds. In the United States, industries exist to supply dry and processed seeds. Our aim is to develop a regeneration system for cowpea as a prerequisite for genetic engineering. Our objective was to examine the in vitro responses of shoot tips to growth regulators. Shoot tips isolated from in vitro-germinated seedlings (`Coronet') were cultured on MS medium containing 2,4-D at 0, 0.01, 0.1, or 1 mg·liter–1 and kinetin at 2.5, 5, 10, or 20 mg·liter–1. Cultures were maintained at 12-hour photoperiods and 24C. Callus, shoots, and roots or combinations thereof developed depending on the treatment. Callus formed on 1 mg 2,4-D/liter, regardless of the kinetin level, but at 0.1 mg 2,4-D/liter and 5 or 10 mg kinetin/liter, shoots also grew. Callus, shoots, and roots developed on 2,4-D lower than 0.1 mg·liter–1. Callus induced on 5 mg kinetin/liter and 0.01 mg 2,4-D/liter regenerated shoots on transfer to 5 mg kinetin/liter and 0.1 mg NAA/liter. This work may assist in the development of a micropropagation system for cowpea.

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J.M. Sherman, K.S. Reddy, S.E. Newman, and J.A. Spencer

The objective of this study was to determine whether tissue culture can be used for studying the blackspot resistance found in some roses. Callus was initiated from leaves, petioles, and stems of resistant and susceptible genotypes. Good callus formation for susceptible roses (hybrid teas) was obtained on a medium containing MS basal salts, vitamins, sucrose, and 8 g/L agar supplemented with 2 mg/L 2,4-D, 1 mg/L NAA, and 0.2 mg/L BA. Callus formation for resistant roses (species roses) was best when the concentrate ions of growth regulators in the medium were halved. Browning in species rose cultures, was decreased with the addition of 0.05 g/L ascorbic acid to the medium followed by incubation in the dark. The subculture calli were inoculated with the fungal conidia and were analyzed for proteins by SDS-PAGE. These protein profiles were compared to those of whole leaf samples. The results are discussed in terms of similarities and differences in the biochemical responses of callus cultures versus whole leaves to the blackspot infection.

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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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Peggy Ozias-Akins and Srini Perera

One cm segments from adventitious roots of sweet potato (Ipomoea batatas (L.) Lam.) will regenerate shoots when cultured on Murashige and Skoog salts and vitamins plus either sucrose (1-3%) or fructose (1-6%). The best source for adventitious roots is sweet potato shoot cultures maintained in Magenta vessels. A low concentration of cytokinin (0.02 mg/liter) promotes shoot formation. Higher levels of cytokinin (0.1-0.5 mg/liter) encourage callus growth. The maximum average number of shoots formed per root segment attained thus far is 0.5. Attempts are being made to increase the frequency of shoot formation. Regeneration of shoots from roots also may be a useful method for obtaining plants from protoplasts of sweet potato. Protoplasts can be isolated from mesophyll tissue and petioles of in vitro grown plants. Plating efficiency of up to 12% routinely can be obtained. Shoot formation directly from callus is sporadic; root formation is more frequent.

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Takashi Ikeda, Yukihiro Fujime, Satoshi Terabayashi, and Shuichi Date

Garlic (Allium sativum L.) calli in vitro were evaluated over a range of salt concentrations and by adding mannitol to culture medium with reduced salt to provide equivalent osmoticum. The water potential of the medium ranged from -0.27 to -0.73 MPa under the various salt and osmotic stress conditions. The percent increase in calli was highest in standard Murashige & Skoog (MS) medium and was reduced when MS salts were reduced but the water potential of medium was adjusted to that of standard MS medium by addition of mannitol. The water potential of callus tissue was similar to that of tissue culture media over a 20-fold range (10% to 200%) of MS concentrations. Turgor of callus tissue was not influenced by any stress conditions. These results indicate that the optimum concentration of salt and water status of medium for formation of garlic calli was provided by standard MS medium.

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Yiqin Ruan and Mark Brand

Combinations of me auxins 2.4-dichlorophenoxyacetic acid (2.4-D). indolebutyric acid (IBA). and naphthaleneacetic acid (NAA), with isopentenylademne (2-iP) were studied in Woody Plant (WP) medium for callus induction and shoot organogenesis from leaves of Rhododendron `Besse Howells' (BH) and `Catawbiense Album' (CA). IBA was more effective than NAA and 2,4-D at inducing shoot organogenesis when combined with 2-iP. Addition of 1 uM IBA and 15 or 30 uM 2-iP to WP medium resulted in the highest percentage of explants producing shoots (90% in BH, 100% in CA), and the greatest number of shoots per explant (18.4 in CA, 10.1 in BH) after 12 weeks of culture. Shoot organogenesis also occurred using 1 uM NAA and 2-iP combinations, but me number of shoots produced was much less than for IBA treatments. 2,4-D and NAA were more productive than IBA for callus induction. Media containing l-10 uM 2,4-D plus 5 uM 2-iP. or 10 uM NAA plus 15 uM 2-iP. were me best for callus production. In studies using thidiazuron instead of 2-iP as the cytokinin, leafy buds and very short shoots developed.

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G.G. Ning and M.Z. Bao

; Olaya et al., 2000 ). For P. mume , an in vitro culture system has been reported for the induction of callus, but plant regeneration was not achieved ( Liu and Chen, 1999 ). Although it is possible to conduct traditional breeding programs in P. mume

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Monte L. Nesbitt, Robert C. Ebel, Douglas Findley, Bryan Wilkins, Floyd Woods, and David Himelrick

Containerized `Owari' satsuma mandarin (Citrus unshiu Marc.) on Poncirus trifoliata `Flying Dragon' rootstock were exposed to one of two acclimation regimes (cold acclimated and unacclimated) and frozen in a computer-controlled freezer to five different low temperatures. Whole plant survival was measured and compared to the results of four leaf and stem injury assays. Acclimating plants in growth chambers at 20 °C day and 10 °C night for 14 days, followed by 15 °C day and 4 °C night for 14 to 21 days resulted in an 81% and 80% increase in leaf and stem survival, respectively, when frozen to a low of -8 °C. Electrolyte leakage and phenolic leakage assays effectively detected changes in percent leaf survival, but the TTC stain assay, using leaf disks, did not. Stem survival was best predicted by the TTC assay, using the phloem as the indicator tissue for survival. Electrolyte leakage and phenolic leakage were also reliable assays for predicting stem survival, although survival percentages were different at the same electrolyte leakage values reported in other studies. The callus growth assay accurately predicted survival for cold acclimated satsuma mandarin stems only. Chemical name used: triphenyl tetrazolium chloride (TTC).