Mango (Mangifera indica L.) somatic embryos representing various developmental stages were subjected to various concentrations of kanamycin in the culture medium. The level of kanamycin necessary for growth inhibition was dependent on the size and stage of the somatic embryos at the time of treatment and the kind of exposure. Growth of proembryos in liquid suspension was arrested at 12.5 μ g·ml-1, while the maturation of later stages of somatic embryos on solid medium was inhibited at 200 μg·m l-1.
Helena Mathews and Richard E. Litz
Lynn M. Long, John E. Preece, Gerald R. Gaffney, J.W. Van Sambeek, and David A. Lightfoot
Genetic transformation studies are aided by use of selection agents, such as antibiotics or herbicides. To determine the level of kanamycin to be used as a selection agent, cotyledonary stage somatic embryos from J. nigra lines J26 and J28, J. nigra × J. hindsii line S11, and J. regia line SU2 were placed on gelrite solidified WPM with 1 g/liter casein hydrolysate and 250 mg/liter cefotaxime and 3% (w/v) sucrose. Dosages for inhibiting secondary embryogenesis were 40 mg/liter kanamycin for J. nigra and J. nigra × J. hindsii and 100 mg/liter for J. regia. For the bialaphos experiments, somatic embryos of J. nigra lines J26 and J28 and J. nigra × J. hindsii line S11 were cultured on gelrite solidified LP medium with 0.5 g/liter casein hydrolysate and 3% (w/v) sucrose. Between 0.1 and 1.0 mg/liter bialaphos, inhibited secondary embryogenesis.
Rodney Serres, Elden Stang, Dennis McCabe, David Russell, Daniel Mahr, and Brent McCown
Genetic transformation of the American cranberry, Vaccinium macrocarpon Ait., was accomplished using electric discharge particle acceleration. Plasmid DNA containing the genes GUS (β-glucuronidase), NPTII (neomycin phosphotransferase II), and BT (Bacillus thuringiensis subsp. kurstaki crystal protein) was introduced into stem sections, derived from in vitro cultures, that had been induced to form adventitious buds. The stage of development of these adventitious buds was critical for efficient initial expression. After exposure to electric discharge particle acceleration, stem sections were cultured on a solid-phase bud-inducing medium containing 300 mg kanamycin/liter. In addition, a thin overlay of 300 mg kanamycin/liter in water was added to inhibit growth of nontransformed cells. Within 7 weeks, green shoots emerged amidst kanamycin-inhibited tissue. No escape (nontransformed) shoots were recovered, and 90% of the transformed shoots were shown through PCR and Southern blot analysis to contain all three introduced genes. GUS expression varied markedly among various transformed plants. Preliminary bioassays for efficacy of the BT gene against the feeding of an economically important lepidopteran cranberry pest have shown no consistently effective control. Potential problems with the expression of the BT and GUS genes are discussed
Jaime A.Teixeira da Silva
Filter paper types significantly affected the growth, development and differentiation of chrysanthemum and tobacco stem thin cell layers (TCLs) from in vitro plantlets. Three different filter paper types, normally with varied uses in plant biology, showed varying morphogenic-altering and antibiotic-buffering capacities. Advantec #2 and Whatman #1 significantly stimulated root, shoot and callus formation while Whatman #3 inhibited them, as compared to TCLs placed directly on agar. Filter paper buffered the phytotoxic effect of antibiotics kanamycin and cefotaxime, substances commonly used in genetic transformation experiments, up to as much as 50%, independent of species or genotype. In both `Lineker' and `Shuhou-no-chikara' chrysanthemum cultivars, Advantec #2 and Whatman #1 filter papers stimulated embryogenesis but in tobacco all three filter paper types significantly reduced embryogenesis and explant survival.
James A. Kapaun and Zong-Ming Cheng
Four aminoglycoside antibiotics were evaluated for their effects on shoot regeneration from leaf explants of Siberian elm (Ulmus pumila L.) seedlings and their potential use as selective agents in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene. Kanamycin at 100 mg·L–1 or higher concentration reduced shoot regeneration, with complete inhibition at 225 mg·L–1, and was considered a suitable selective agent. Neomycin completely inhibited shoot regeneration at 450 mg·L–1, but all explants remained green; therefore, it may also be used as a selective agent. Geneticin significantly inhibited shoot formation at 1 mg·L–1 and completely killed the explants at 4 mg·L–1 after 1 week. Geneticin was too toxic for direct selection, but may be useful in a delayed selection scheme or for confirmation of transformation. Paromomycin was least effective in inhibiting shoot formation; 13% of explants still regenerated shoots on the medium with the highest concentration tested (400 mg·L–1). Both neomycin and paromomycin precipitated in media containing Phytagel as a gelling agent if antibiotic stock solutions were added to the medium without adjusting their pH. Precipitation was prevented by adjusting the pH of the stock solutions from 6.2 (neomycin) or 6.9 (paromomycin) to above 9, or by using agar as a gelling agent. The precipitation was not affected by the concentrations of salts in the media.
Wenhao Dai, Victoria Magnusson, and Chris Johnson
.) containing the npt II gene encoding for neomycin phosphortransferase and the uid A coding for β-glucuronidase (GUS) ( Fig. 1 ), was grown overnight in LB (Luria-Bertani) medium with 100 mg·L −1 kanamycin at 28 °C in a shaker at 150 rpm. Cells were
Wenhao Dai, Yuanjie Su, Hongxia Wang, and Ceilo Castillo
photoperiod. Plant transformation. An efficient shoot regeneration system of these two Buddleia cultivars was developed by Dai and Castillo (2007 ). Previous experiments determined that kanamycin at 40 mg·L −1 completely inhibited shoot regeneration from
Kathryn Kamo and Bong Hee Han
auxins and kanamycin. Successful transformation of ‘Snow Queen’ was achieved using kanamycin for selection of putative transformants ( Cohen et al., 2004 ), but our ‘Nellie White’ callus and plants cannot be selected using kanamycin because both grow on
Yu Liu, Miao He, Fengli Dong, Yingjie Cai, Wenjie Gao, Yunwei Zhou, He Huang, and Silan Dai
genetic transformation of C. grandiflora refers to the method of Liu (2015) . After kanamycin sensitivity tests, selection for plant rooting pressure, precultivation, A. tumefaciens infection, cocultivation, and delayed cultivation, the resistant
Joseph C. Kuhl, Kelly Zarka, Joseph Coombs, William W. Kirk, and David S. Douches
construct called pSPUD69 ( Fig. 1 ). Agrobacterium tumefaciens (Smith & Towns.) Conn.-mediated transformations using a kanamycin marker were conducted according to Douches et al. (1998) . The explants were prepared for transformation by cutting internodes