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Aaron Brown and Harrison G. Hughes

Callus induction and regeneration of alkaligrass (Puccinellia distans) was developed in our laboratory for use in transformation studies of turfgrass. Particle bombardment of the embryogenic callus is being evaluated using a helium particle inflow gun constructed at Colorado State Univ., according to the design of Philippe et al. (Ohio State Univ., 1993). Its utility in delivering DNA to plant cells is being tested by measuring the frequency of transient gene expression of a reporter gene (GUS pBI121) in embryogenic callus of alkaligrass. Varying pressure of helium and the distance of the calli in the chamber are also being evaluated for efficiency in transformation.

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F.A. Hammerschlag

A detached-leaf bioassay was used to evaluate peach [Prunus persica (L.) Batsch] regenerants derived from zygotic embryo callus cultures of cultivars Sunhigh (susceptible to leaf spot) and Redhaven (moderately resistant to leaf spot) for resistance to Xanthomonas campestris pv. pruni [(E.F. Sm.) Dews], the causal agent of bacterial leaf spot. Regenerants obtained from calli produced on two `Sunhigh' embryos, #61 and #156, and on three `Redhaven' embryos were evaluated. Sixty-four percent of the regenerants derived from `Sunhigh' embryo #156 and 13% of the regenerants derived from `Sunhigh' embryo #61 demonstrated significantly greater spot resistance than `Sunhigh'. Regenerants with resistance greater than `Redhaven' were also obtained from both `Sunhigh' embryos. The frequency of variation in the `Sunhigh' seedling population, with respect to the response to bacterial leaf spot, was not so great as that exhibited by the regenerants derived from `Sunhigh' embryo #156. None of the `Redhaven' seedlings or any of the regenerants derived from `Redhaven' embryos were more resistant than `Redhaven'. These studies suggest that the frequency of somaclonal variation is genetically determined and that screening for somaclonal variation may be a feasible approach to obtaining leaf spot-resistant peach plants.

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Wanmei Jin, Jing Dong, Yuanlei Hu, Zhongping Lin, Xuefeng Xu, and Zhenhai Han

cocultivated with the Agrobacterium strain was 4480. Callus appeared 8 to 10 d after inoculation and 100% calli were produced and 35.6% regenerative calli were induced 28 d after cultured in the selective media. Transformants were generated from green or

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Kimberly A. Pickens, (Max) Zong-Ming Cheng, and Stephen A. Kania

Poinsettia, Euphorbia pulcherrima, is an important holiday symbol and is the number one flowering potted plant in the United States. The technique of chromosome doubling has been utilized to increase size of flowers, stems, and leaves of many species, and has been used in poinsettia breeding to obtain new cultivars. Application of colchicine or oryzalin to in vitro tissues may be used to enlarge the inflorescences and brackets and reduce the height of `Winter Rose'™ poinsettias, reduce the likelihood of chimeric tetraploids, and provide a rapid means for producing many tetraploid plants. The purpose of this research was to evaluate the effect of colchicine and oryzalin on callus and adventitious shoot formation of `Winter Rose'™ poinsettia with in vitro grown leaf tissues and its potential for tetraploid induction. In vitro grown leaf midvein sections were placed on various media supplemented with either colchicine or oryzalin at various concentrations for 1–4 days. Colchicine was least damaging to leaf tissues at concentrations of 0.25 or 250.4 μm. A large amount of callus, as well as adventitious shoots, were produced. Regenerated shoots were found to be diploid, determined by flow cytometry. On media with oryzalin (28.9–144 μm), leaf tissues produced callus, but not adventitious shoots. Calluses produced on oryzalin-containing media were tested using the flow cytometer and were found to be diploid.

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Ronald W. Moore, K.M. Eskridge, P.E. Read, and T.P. Riordan

The concept that greater callus mass will induce competence was investigated. The second most immature nodal segments were removed from heavily fertigatcd greenhouse grown plants. Shoots initiated from those nodes were only cut back to one-third their total length. They were subjected to the following treatments: (1) dicamba from 1μM to 5μM in increments of 1.0; (2) B5 medium salt concentrations from 1/3x to 5/3x in increments of 1/3; (3) sucrose levels from 2% to 10% in increments of 2; (4) casein hydrolysate from 0 to 200mg/l in increments of 50. The experiment consisted of twenty-five different treatment combinations in a central composite rotatable second order design. Explants were placed in continuous cool white fluorescent light at 26°C.

Dicamba, B5 salts, and sucrose had significant effects on callus mass (p<.12), while casein hydrolysate had no notable effects on callus mass (p ≥ .57). It was determined that optimum response occurred at 5/3x concentration of B5 salts, 10% sucrose, and 5.0μM dicamba. White, compact calli were observed in treatment combinations that yielded callus fresh weights of two-hundred milligrams or higher.

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Lisa C. Berg and Henry R. Owen

Nicotiana tabacum callus growth (fresh weight) was measured after culture in the light (16-hour photoperiod) or in darkness for four different culture media, differing in iron chelate type or concentration. All media contained MS basal medium supplemented with 30 g·L–1 sucrose, 2 mg·L–1 IAA, 0.2 mg·L–1 KIN, and 7 g·L–1 agar, pH 5.8. Three of the media contained iron-metalosate (Albion Laboratories), an organic iron chelate, at 100, 200, and 400 micromolar concentrations, and the fourth medium contained 100 μm Fe-EDTA. Twenty-five culture tubes were prepared for each of the 4 different media concentrations and 2 light treatments (8 treatments total). A 1-cm3 callus explant was used for each treatment and cultured for 56 days at 20°C. About 20-fold increases in callus fresh weight were observed for cultures incubated in light or in darkness. In addition, callus growth was not significantly affected by iron chelate type, suggesting the potential utility of this organic chelator in tissue culture media to alleviate potential problems of light-induced EDTA instability and subsequent IAA inactivation. These cultures are being maintained to examine the influence of iron chelate type on organogenesis.

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Carol D. Robacker

Nodes from in vitro grown shoots of `Regale' and `Triumph muscadine grape (Vitis rotundifolia) were cultured in 25 × 95-mm glass vials, 25 × 150-mm glass culture tubes, 55 × 70-mm glass baby food jars and 100 × 25-mm plastic petri dishes. Culture medium consisted of Murashige and Skoog salts and vitamins, 80 mg/l adenine sulphate, 170 mg/l sodium phosphate, 2 mg/l BA and 8 g/l agar. Amount of medium dispensed was 8 ml per vial, 18 ml per culture tube and jar, and 70 ml per petri dish. Containers were covered with clear plastic lids, sealed with parafilm, and placed under fluorescent lights for six weeks. The number of shoots per explant that formed in petri dishes was three to four times greater than those formed on explants in vials, tubes and jars. However, the number of nodes per shoot were fewer in dishes than in the other containers. Callus formation was excessive in jars to the detriment of shoot production. Vials and tubes had small amounts of callus, while little or no callus was observed in dishes.

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Rajendra Maurya*, Nathu Ram Godara*, and Ram C. Yadav*

Influence of culture media and hormone concentrations on plant regeneration from rose (Rosa hybrida L. cv. Raktagandha) leaf segments were investigated. Leaves were excised from healthy, well-grown and mature plants. Leaf segments (4-5 mm long) were sectioned and cultured on Murashige and Skoog (1962) medium containing different concentration of growth hormones. Callus formation was most prolific (97.09%) on MS medium containing MS basal salts + 0.5 mg·L-1 BAP + 2.0 mg·L-1 2,4-D. maximum (56.67%) organogenesis or shoot differentiation was achieved on MS modified medium supplemented with 1.0 mg L-1 BAP + 0.1 mg·L-1 NAA + 10.0 mg·L-1 Adenine Sulphate. The highest percentage (93.73%) of in-vitro rooting was observed in half-strength MS basal medium containing 0.5 mg·L-1 IBA. Rooted plants were transferred in to sterilized potting mixture and grown in a greenhouse.

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W.A. Mackay, T.J Ng, and F.A. Hammerschlag

Cucumis melo L. (muskmelon) is susceptible to Myrothecium roridum at all stages of growth with no known source of resistance. Screening of regenerants from long-term cotyledonary-derived callus cultures of muskmelon cv. Hales Best (tolerant), Iroquois (susceptible), and Perlita (intermediate) was carried out to determine if novel plants with increased levels of resistance could be obtained. A detached-leaf bioassay was used to screen greenhouse-grown regenerants and seedlings of the three cultivars. Resistance was determined by measuring necrotic lesion diameter and chlorotic plus necrotic lesion diameter. No change in the level of resistance to M. roridum has thus far been observed. Thus, screening for somaclonal variation may not be aviable approach to recover resistance in muskmelon to M. roridum.

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Maurus V. Brown, James N. Moore, and Fens H. Huang

`Saturn', `Mars', and `Reliance' were compared based on their different Vitis vinifera and V. labrusca compositions. Disks (10 mm) from young leaves were placed abaxial side down on a standard media containing NAA or 2,4-D at 0.0, 1.0, and 2.0 mg/L with BAP at 0.0, 0.1, and 0.2 mg/L. Each treatment was replicated in 10 culture tubes and incubated at 25 ± 1C under cool-white fluorescent light for 10h photoperiods. Calli were compared by size, color, and occurrence of morphogenesis. NAA generally produced a larger callus by cultivar than 2,4-D. A greater quantity of callus was generally produced with the increase of the V. labrusca component. Callus produced on 2,4-D medium was round, compact and light to dark green in color. However, callus produced on NAA medium was amorphous, friable, and ranged in colors. Rooting occurred on some calli produced on NAA media.