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Servet Kefi, Paul E. Read, Alexander Pavlista, and Stephen D. Kachman

To determine the influence of gibberellic acid (GA3) and 6-furfuryl aminopurine (kinetin) concentrations alone and in combinations on in vitro tuberization of potato, nine treatments consisting of combinations of gibberellic acid and kinetin at three levels of concentration (0, 2, and 5 mg·liter–1) were included in Murashige and Skoog medium supplemented with 6% sucrose. Four single nodes of in vitro plantlets from Solanum tuberosum L. cultivar Atlantic were placed into each magenta box. All magenta boxes were arranged in a randomized complete box design with five replications and cultured under a short photoperiod condition (8 h light/16 h dark). Gibberellic acid strongly inhibited tuberization when used alone or with kinetin, whereas kinetin induced tuberization at both 2 and 5 mg·liter–1. Although tuberization was initiated in the absence of kinetin because of the high concentration of sucrose and short photoperiod, the presence of kinetin accelerated the in vitro tuberization process of potato.

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Azza A. Tawfik, Susan L. Cuppett, and Paul E. Read

Shoot tips (7 to 10 mm long) of rosemary plant (Rosmarinus officinalis L. 'Lockwood de forest') were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) (0, 0.5, 1.0, 1.5, and 2 mg/l) alone or with 3-indole acetic acid (IAA) at 0.5 mg/l. The effect of TDZ and IAA on the proliferation of rosemary shoot tips has been reported in a previous meeting. Here, we report on the effect of TDZ and IAA on the monoterpene constituents identified in the oil of rosemary plants propagated in vitro. The proliferated explants were soaked in hexane as a solvent, then the extractions were used for monoterpene analysis using GC/MS. A significant interaction of TDZ by IAA was found on most of the oil components identified. The highest levels of 1,8-cineole and borneol were obtained at 0.5 mg TDZ/l alone, while the highest level of camphor was obtained at 0.5 mg TDZ/l plus 0.5 mg IAA/l. The highest level of bornyl acetate was at 2 mg TDZ/l.

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

Few studies on embryogenesis in common bean (Phaseolus vulgaris L.) have been reported and only the early stages of somatic embryogenesis were observed. Dry seeds from two common bean lines were germinated in darkness on L-6 medium containing 4% sucrose, 0.2 g casein hydrolysate /liter and 2.0 g phytagel /liter. The medium for seed germination was supplemented with 0, 2, 4 or 6μM forchlorfenuron (CPPU). Explants from cotyledonary leaves, petioles, hypocotyls and shoot apices were prepared from 14 day-old seedlings. Callus was derived from explant cultures incubated in darkness at 26C on the medium containing 4 μM 2,4-D and 1 μM Kinetin. The callus was transferred after 4 weeks into 125 ml Erlenmeyer flasks containing 50 ml liquid medium and placed on a gyrotary shaker (120 rpm) under cool-white light (12 μmol.m-2.s-1). The liquid medium was used with 2, 4 or 6 μM of 2,4-D alone or with zeatin supplements at relative concentrations of 0.25 and 0.5. Up to 200 somatic embryos from 40 to 50 mg callus inoculations were induced after 4 to 5 weeks. Callus derived from seedlings grown on CPPU-containing medium gave more repetitive somatic embryos. Cotyledonary stage embryos with clear bipolar structure were observed only from callus derived from seedlings grown on CPPU when transferred to suspension cultures containing 2,4-D and zeatin. All somatic embryos differentiated strong roots and some developed leaf-like structures on conversion medium.

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Azza A. Tawfik, Paul E. Read, and Susan L. Cuppett

Callus of Rosmarinus officinalis L. 'Lockwood de forest' was induced from stem segments (3 mm long) using different concentrations of thidiazuron (TDZ). The original stem segments used as explants were found to have a higher level of linalool than was found for leaf segments. Linalool is one of the monoterpenes identified in rosemary plants and it has a pleasant aroma. TDZ has a significant effect on callus formation and callus texture. The callus formed was light green to yellow and/or had some meristimatic dark green cells. TDZ had a significant linear effect on the callus fresh weight. The meristimatic green cells formed on all calli except those proliferated on the lowest concentration of TDZ (0.5 mg/l). No callus was induced from stem segments cultured on TDZ-free medium. The fresh calli from other treatments were soaked in hexane as a solvent for monoterpene analysis using GC/MS. No monoterpenes could be detected in the callus induced on the medium containing the lowest concentration of TDZ. Comparing to the stem segments taken from the parent plants only 4 of 10 monoterpenes identified were found in the callus: α-pinene, β-pinene, 1,8-cineole, and camphor.

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

Rosemary Rosmarinus officinalis is a member of the Lamiaceae. Rosmarinic acid (RA) is a very strong antioxidant produced in the chloroplast, and used to protect plant tissues against oxidative stress. A number of investigations showed that the sucrose concentration in the callus growing medium greatly influenced the production of secondary metabolites of the phenylpropanoid pathway such as RA. The aim of this study was to test the effect of elevated sucrose concentrations (2%, 3%, 4%, 5%, and 6% sucrose) and the effect of light and dark treatments on the production of RA in the callus of five different genotypes. The genotypes were Majorca, Rosmarinus officinalis, Pine Scented, Madeline Hill, and APR. It was found that the dark treatment produces more RA than the light treatment in all genotypes, and in all sucrose concentrations. The RA concentration increased with increasing the sucrose concentration from 2%—reaching the highest concentration at 4% and 5% in most genotypes. The RA concentration declined again at 6% sucrose in all genotypes. We concluded that for the extraction of RA from rosemary callus it is preferred to be produced in the dark—this will save energy and will produce more RA than the light treatment. Also it is preferred to use sucrose concentration at 4% for genotypes Rosmarinus officinalis, Pine Scented, and APR; and 3% sucrose for genotype Madeline Hill in the dark condition. While for the light condition, it is preferred to use 5% sucrose with genotypes Majorca, Rosmarinus officinalis, Pine Scented, and Madeline Hill; and 4% sucrose for genotype APR.

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Hany M. El-Naggar, Paul E. Read, and Ayed Al-Abdallat

Phenylalanine ammonia-lyase (PAL) enzyme is the most extensively studied enzyme in the phenylpropanoid pathway. Studies on the biosynthesis of rosmarinic acid (RA) showed that the PAL enzyme catalyzes the initial step of the phenylpropanoid pathway. The increase in RA content in plant tissues in vitro coincided with the increase in PAL activity. The aim of this study was to investigate the activity of the gene responsible for the production of the PAL enzyme in the five rosemary genotypes; this will give more understanding about the accumulation of rosmarinic acid in the five rosemary genotypes. The genotypes were Majorca, Rosmarinus officinalis, Pine Scented, Madeline Hill and APR. Northern blot hybridization between the PAL gene primer and the five genotypes' cDNA showed bands at 300 bp in all the five genotypes for the PAL gene. The expression of the PAL gene was high in genotypes Majorca, Rosmarinus officinalis, and Madeline Hill, while the expression was low in genotypes Pine Scented and APR. It was expected that the genotypes having the highest PAL gene expression will produce the highest amount of RA, but the highest genotype in PAL gene expression Madeline Hill had the lowest RA production in their leaves. This could occur due to the tissue specific regulation inside plant tissues. Inside the callus tissues, where the specific tissue regulation no longer exists, the RA was produced in repetitively large amounts in genotypes with high PAL gene expression.

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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.

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Virginia Miller-Roether, Paul E. Read, and Erika Szendrak

The American Chestnut Foundation (ACF) has conducted a breeding program aimed at developing blight-resistant chestnut trees exhibiting the phenotype of American Chestnut (Castanea dentata). We developed a protocol for in vitro micropropagation and multiplication of candidate blight-resistant plants from the ACF breeding program. The protocol included forcing dormant shoots to budbreak, culture establishment, shoot multiplication, inducing a functional root system on the microcuttings produced by this system and establishment of autotrophic plants. Because Castanea spp. is recalcitrant to rooting, a unique bilayer method of rooting was developed. The unique bilayer consisted of a clear basal medium of 50% DKW and 50% WPM (Long and Preece), with a continuous level of 0.01 mg IBA/L and 0.2 mg BA/L. The clear basal medium was over-laid with an opaque layer. Rooting response occurred for 27 of the 31 genotypes at various frequencies. Rooted plantlets were planted in 50% peat: 50% perlite in order to become autotrophic and acclimated. Acclimated trees were planted in 10″ × 2″ Deepots® and placed in the greenhouse. These trees exhibited a very vigorous functional root system. Acclimated trees were hardened off, placed in cold storage (≈4-5 °C) for 5 months. All trees placed in cold storage broke dormancy for spring growth and ≈100 trees were sent to ACF for planting into field trials.

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

The leaf reaction of the Phaseolus vulgaris L. germplasm—UNECA (M6 mutant derived from the cultivar Chimbolito, Costa Rica), `Chimbolito', BAC-6 (Brazil), XAN-159 (Centro Internacional de Agricultura Tropical, Cali, Colombia), and `PC-50' (Domican Republic)—to Xanthomonas campestris pv. phaseoli strain V4S1 (Dominican Republic) were determined in two replicated trials conducted in a greenhouse in Lincoln, Neb. (Feb.–Mar. and July–Aug. 1993). `PC-50' and `Chimbolito' were susceptible to Xcp strain V4S1 in both tests. UNECA, BAC-6, and XAN-159 had similar levels of resistance to Xcp in the July to August trial. However, in the February to March trial, the resistance of UNECA was greater than that of BAC-6 but less than that of XAN-159.

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Shuizhang Fei, Paul E. Read, and Terrance P. Riordan

The use of buffalograss [Buchloe dactyloides (Nutt.) Engelm] in home lawns and golf courses has been increasing because of its drought resistance and low growth habit. In vitro regeneration of buffalograss at a high frequency may provide an effective tool to introduce new variation for breeding use. The positive effects of AgNO3 on friable embryogenic callus production and regeneration efficiency is well documented in maize. In order to determine if AgNO3 has the same effect on buffalograss, two vegetatively propagated cultivars, a female `609' and a male `45-3' were tested at three different concentrations of AgNO3 at 5, 10, and 15 mg·L–1 using immature inflorescences as explants. Murashige and Skoog medium supplemented with 2 mg 2,4-D/L was employed as the control medium. Medium containing AgNO3 significantly promoted the production of friable callus for `45-3' with the highest percentage achieved at 10 mg AgNO3/L. AgNO3 medium led to production of significantly larger calli than found for the control. However, no difference was detected among 5, 10, and 15 mg AgNO3/L with regard to the callus formation ability and the size of callus initiated on these three treatments. Calli were then transferred to MS medium supplemented with BA at 0.1, 0.5 or 1.0 mg·L–1 to induce shoot formation. BA at 0.5 mg·L–1 gave the best differentiation response. Calli formed in the absence of AgNO3 produced more shoots per callus, but more calli were produced in the presence of AgNO3, and the overall regeneration efficiency was much higher with AgNO3 at 10 mg·L–1. In contrast, AgNO3 showed no promotive effect on callus production and regeneration for `609'.