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J. Jaramillo and W.L. Summers

Anthers from. three tomato cultivars, `L-680A', `Ailsa Craig', and `Licato', were plated on DBM1 medium solidified with one of four solidifying agents, Bacto-agar, Gelrite, Noble agar, or Phytagar, to evaluate their ability to promote initiation and growth of tomato anther callus. The optimum concentration of each solidifying agent was compared with a liquid control. Optimum levels of the various solidifying agents were (in g·liter-1) Phytagar, 5; Gelrite, 3; Noble agar, 6 and Bacto-agar, 8. Both the number and diameter of calluses were affected by type of solidifying agent and anther genotype. Significant interactions were also found between tomato cultivars and solidifying agent. Noble agar produced good results with `L-680A' and `Ailsa Craig', but not with `Licato'. Bacto-agar reduced the number and size of callus by 38% when compared with the liquid treatment and by 42% when compared with the best agar treatment (Noble agar).

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Jing-Tian Ling, Nobumasa Nito, Masao Iwamasa, and Hisato Kunitake

Embryos were obtained from unfertilized and undeveloped seeds of satsuma (Citrus unshiu Marc.) cultured on a modified Murashige and Tucker (MT) medium. Embryogenic callus was induced from the hypocotyl region of the embryos. The callus was successfully maintained through subculturing on MT medium with 185 μm ade-nine, 2.8 μm GA3 and 400 mg malt extract/liter, solidified with Gelrite. Somatic embryogenesis occurred from callus subculture on medium containing 50 g lactose/liter and in the absence of plant growth regulators. Somatic embryos developed into plants on medium with sucrose and GA3. Protoplasts isolated from this callus produced somatic embryos through colony formation: subsequently, normal, entire plants were regenerated.

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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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Mahmoud B. Arif and Houchang Khatamian

Friable callus from leaf disks of Rosa hybrida `Tiffany' was initiated within two weeks under dark conditions and 25°C on Murashige and Skoog (MS) medium supplemented with 4 mg.liter-1 2,4-D. Callus was then transferred into MS medium containing 3 mg.liter-1 2,4-D. Within four weeks, rhizogenesis occurred on the callus surface.

The rhizogenic calllus was subculture on MS medium plus 3 mg.liter-1 2,4-D every 4-6 weeks. Within six months from initial culture, somatic embryos were developed on the aging callus in darkness.

Transfer of the aging callus with somatic embryos into 1/2 MS medium containing 1 mg.liter-1 kinetin and maintaining it under 46 μE m-2s-1 light for 16 hrs. resulted in greening of the somatic embryos.

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Mahmoud B. Arif and Houchang Khatamian

Friable callus from leaf disks of Rosa hybrida `Tiffany' was initiated within two weeks under dark conditions and 25°C on Murashige and Skoog (MS) medium supplemented with 4 mg.liter-1 2,4-D. Callus was then transferred into MS medium containing 3 mg.liter-1 2,4-D. Within four weeks, rhizogenesis occurred on the callus surface.

The rhizogenic calllus was subculture on MS medium plus 3 mg.liter-1 2,4-D every 4-6 weeks. Within six months from initial culture, somatic embryos were developed on the aging callus in darkness.

Transfer of the aging callus with somatic embryos into 1/2 MS medium containing 1 mg.liter-1 kinetin and maintaining it under 46 μE m-2s-1 light for 16 hrs. resulted in greening of the somatic embryos.

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Michele Warmund, Bruce Barritt, and Karen Schaffer

`Mark' rootstock is a relatively new dwarfing rootstock that induces precocity in apple trees. While `Mark' has desirable horticultural characteristics, it has been difficult to propagate in some areas of the United States. To determine the optimum budding date at two climatically diverse locations, `Jonagold' buds were chip-budded onto `Mark' rootstock on 20 July, 10 Aug., 31 Aug. and 21 Sept. 1989 at Atlas, Illinois and Wenatchee, Washington. Prior to budbreak, unions were sampled from each budding date and the callus, bud plate and rootstock were measured and photographed. Trees budded and grown in Illinois had more callus growth than those budded in Washington. In Illinois, the callus of trees budded on 20 July averaged 3.2 mm., whereas those budded on 21 Sept. averaged 1.0 mm. Trees grown in Washington had 0.4 mm of callus at both budding dates. Callus growth will be correlated with union compatibility and strength in Nov. 1990.

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C.K. Kim, J.Y. Oh, J.D. Chung, A.M. Burrell, and D.H. Byrne

Somatic embryogenesis was initiated from in vitro-grown leaf explants of rose using an induction period of 4 weeks on MS basal medium supplemented with auxin followed by several subcultures on MS basal medium with cytokinin. `4th of July' showed the highest regeneration frequency (24.4%) on 5.3 μm NAA followed by culture on medium containing 18.2 μm zeatin. `Tournament of Roses' produced somatic embryos when cultured for 4 weeks on medium containing dicamba, 2.3 μm followed by three subcultures on medium containing 18.2 μm zeatin. Embryogenic callus matured on MS media containing 0.5 μm NAA, 6.8 μm zeatin, and 2.9 μm GA3. Long-term cultures were established for both cultivars. Somatic embryos germinated on MS medium containing IBA and BA. Silver nitrate (58.8 μm) enhanced shoot formation and germination of somatic embryos. Plants derived from somatic embryos were acclimatized and successfully established in the greenhouse.

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J. Jaramillo and W.L. Summers

Three tomato (Lycopersicon esculentum Mill.) cultivars were studied to determine the effect of nine dark-light regimes on anther callus initiation and growth. Prophase I (leptotene) stage anthers of `L-680A', `Licato', and `Ailsa Craig' were plated on Doy's basal medium 1 and provided with 2 to 10 weeks of dark treatment. After each dark period, each plate was transferred to a 16-hour photoperiod for the duration of the 10-week experiment. At this time, the number of anthers producing calli and the diameter of the calli produced were recorded. Callus number and diameter increased as the dark period duration increased. Callus diameter peaked at 8 weeks of dark treatment for `Ailsa Craig' and `Licato', whereas `L-680A' calli continued to grow over the entire 10-week dark treatment. Although the number and size of callus may continue to increase past 8 weeks of dark incubation, callus quality and appearance decreased noticeably during this period. For each additional week of dark period exposure, 7% more of the plated anthers produce callus, and callus diameter increases by 0.27 mm (12% of total growth). -

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Guochen Yang and Marihelen Kamp-Glass

The success of genetic transformation depends on the efficiency and rehability of in vitro shoot regeneration. This research was pursued to investigate how different plant growth regulators influence alfalfa callus initiation and development, thus to establish a foundation for further development of an efficient shoot organogenesis protocol for the genetic transformation system. BA, zeatin, and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were evaluated for callus initiation and development. BA at 1 or 5 mg·liter–1, or zeatin at 5 mg·liter–1 promoted callus regeneration and further development toward shoot organogenesis. However, 2,4,5-T at 1 mg·liter–1 enhanced only callus production. These results can and will be used for further development of a shoot regeneration protocol to assist alfalfa genetic transformation.

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Christopher M. Long, Colleen A. Mulinix, and Amy F. Iezzoni

Microspore-derived callus cultures were obtained by anther culture of `Emperor Francis' sweet cherry (Prunus avium L.). Branches were removed from the field in January and March and forced in the laboratory. When the microspores reached the uninucleate stage, anthers were placed on modified Quoirin and Lepoivre liquid culture medium containing 4.4 μm BA and 4.5 μm 2,4-D. After ≈60 days, callus that emerged from the anthers was placed on woody plant medium supplemented with 1 μm 2,4-D and 3 μm 2iP and routinely transferred. The resulting 270 callus cultures were screened for two allozymes heterozygous in `Emperor Francis', Pgi-2 and 6-Pgd-1. Of the 270 callus cultures, 154 expressed only one allele each for Pgi-2 and 6-Pgd-1; thus, they were considered microspore-derived. The microspore-derived callus cultures can be used as a linkage mapping population. Chemical names used: 6-benzyladenine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); N6-(2-isopentenyl)-adenine (2iP).