Most bacteria isolated from persistently contaminated micropropagated mint plants were Gram-negative rods identified as xanthomonads, pseudomonads, and agrobacteria based on their cultural characteristics. A few Gram-positive, non-sporeforming bacteria were also found. Inhibition of bacterial growth by gentamicin and streptomycin was greater at pH 6.5 and pH 7.5 than at pH 5.5. Inhibition by rifampicin and Timentin was less affected by pH change. Pseudomonads were uniformly resistant to Timentin at all pH's and at levels up to 1000 μg/ml. Streptomycin at 500 μg/ml was bactericidal for the pseudomonads and Gram-positive bacteria while 1000 μg/ml was required to kill xanthomonads and agrobacteria. Minimal bactericidal concentrations for gentamicin varied widely, even within groups, and ranged from 10 μg/ml to >80 μg/ml for agrobacteria. These results emphasize a need to acquire basic information about the identities and antibiotic susceptibilities of microbial contaminants before attempting treatment of infected plant cultures.
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.
Some antibiotics mimic plant hormones on cell growth and plant regeneration. Cefotaxime and carbenicillin were tested in American elm for induction of embryogenesis from cotyledonary explants, which normally show organogenesis. Cotyledons from 1-week-old in vitro germinated seedlings were placed on a shoot regeneration medium (a modified MS medium containing 15 μ M BA, B5 vitamins and 0.3% gelrite) with various levels of cefotaxime and carbenicillin. One hundred percent of explants showed embryogenesis in the medium supplemented with 125 μg/ml cefotaxime; 75% explants regenerated somatic embryos in medium with 500 μg/mg carbenicillin; and only 50% explants produced somatic embryos in the medium with both of these antibiotics. In control medium without antibiotics, 100% explants regenerated shoots, instead of somatic embryos. Further studies are necessary to determine the nature of these antibiotics on shifting developmental pathways and their stimulatory effect on embryogenesis from American elm cotyledons.
contamination ( Kunisaki, 1980 ). Internal contamination can be minimized or eliminated using antibiotics incorporated into the culture media ( Kneifel and Leonhardt, 1992 ) or with pretreatment of explants before in vitro culture ( Kritzinger et al., 1998
Regeneration from apple (Malus × domestica Borkh.) M.26 leaf tissue was completely inhibited by (μg·ml-1) 1 geneticin, 5 kanamycin, 10 to 25 paromomycin, and 100 neomycin. nptII-transgenic M.26 had an increased tolerance to all four of the antibiotics tested, with inhibition of regeneration occurring at (μg·ml-l) 2.5 geneticin, 100 kanamycin, 375 paromomycin, and 375 neomycin. Paromomycin (100 to 250 μg·ml-l) and neomycin (250 μg·ml-1) significantly increased the amount of regeneration from nptII-transgenic M.26 apple leaf tissue. p35SGUS-INT, a plasmid with a chimeric b -glucuronidase gene containing a plant intron, was useful for studying the early events of apple transformation by eliminating GUS expression from Agrobacterium tumefaciens. It was used to determine that the optimal aminoglycoside concentrations for the selection of nptII-transgenic M.26 cells were (μg·ml-1) 2.5 to 16 kanamycin, 63 to 100 neomycin, and 25 to 63 paromomycin. Geneticin was unsuitable as a selective agent.
Pseudomonas cepacia LT412W reduced green mold on lemons caused by Penicillium digitatum. It produces phenylpyrrole antibiotics which cause inhibition zones in co-culture with the pathogen. Their role in control of the disease was investigated. Mutagenesis of P. cepacia (rifampicin resistant) was performed by mating it with E. coli S-17 pSUP1021 (kanamycin resistant), which carries the transposon Tn5. Transconjugate selection and screening for absence of inhibition zones identified a stable mutant. Growth of parent and mutant were comparable. When the mutant was co-cultured with the pathogen on lemon albedo agar, no inhibition zone appeared. Similar co-culture on potato dextrose agar with tryptophan (0.05 g/L), a precursor of phenylpyrroles, did not induce inhibition zones. This suggests the mutation is not in tryptophan biosynthesis. Parent and mutant were assayed for phenylpyrroles. They were cultured in nutrient broth, centrifuged, and the cells extracted with acetone. The extract was dried and dissolved in chloroform. It was spotted on nano-SIL Cl8 TLC plates, run one hour (methanol:acetonitrile:water, 1:1:1), dried, developed with sulfanilic acid, and observed under UV light. The relative mobility of spots from extracts of the parent matched phenylpyrroles, whereas the mutant produced none. Control of decay by the mutant and parent were equal, suggesting no role for phenylpyrroles in suppression of the disease.
Ten bacteria were isolated from the in vitro shoot cultures of different tansy (Tanacetum vulgare) genotypes. All isolates were Gram-negative. Five isolates belonged to Enterobacteriaceae, three isolates were strains of fluorescent Pseudomonas, and two isolates were strains of other aerobic bacteria. The combined treatment with gentamicin (50 mg·L–1) and rifampicin (25 mg·L–1) prevented the growth of all bacteria, whereas none of the antibiotics (ampicillin, cefotaxime, rifampicin, gentamicin, or streptomycin) controlled all bacteria when used alone. The antibiotics ranged based on their increasing adverse effects on shoot cultures as follows: rifampicin, cefotaxime, and gentamicin. Increased antibiotic concentration reduced the initiation and growth rates of shoots, roots and calli with some exceptions. Low concentrations of gentamicin and cefotaxime both increased the growth rate and shoot number per plant. Rifampicin stimulated the root growth of some of the biotypes tested. The growth rate of calli increased significantly in the presence of both gentamicin (25 mg·L–1) and rifampicin (25 mg·L–1) in the growth media, whereas the use of either antibiotic alone reduced the calli growth relative to the control. Viability of tansy protoplasts was enhanced by rifampicin and to a lesser extend by gentamicin and cefotaxime. The growth of bacteria isolated from tansy tissue culture can be prevented with a combined gentamicin and rifampicin treatment. The growth rate of plants decreased slightly when grown in the media supplemented with the same antibiotic combination. However, the growth retardation was not permanent, and the plants recovered and grew vigorously when transferred to antibiotic free medium.
A range of antibiotics was evaluated for their effect on eliminating Agrobacterium tumefaciens supervirulent strain EHA101(pEHA101) from leaf explants of `Royal Gala' apple (Malus domestica Borkh) and on regeneration. After long-term (38 days) exposure to 100-μg–ml–1 concentrations of either cefotaxime (cef), carbenicillin (carb), mefoxin (mef), or combinations of these antibiotics, only on carb or carb with mef was regeneration not inhibited. None of the above antibiotics or antibiotic combinations eliminated A. tumefaciens from leaf explants. Short-term (1-18 hours), vacuum infiltration with 500- to 1000-μg–ml–1 concentrations of either of the above antibiotics did not inhibit regeneration, but did not eliminate A. tumefaciens from leaf explants. After a 30-min vacuum infiltration with a 2000-μg–ml–1 concentration of either cef, carb, or mef, only cef reduced the number of leaf explants with A. tumefaciens.
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.
A range of antibiotics and short-term exposure to an acidified (pH 3.0) medium were evaluated for their effects on eliminating Agrobacterium tumefaciens, supervirulent strain EHA101 (pEHA101/pGT100), from leaf explants of `Royal Gala' apple (Malus ×domestica Borkh.) and on shoot regeneration. Exposure of leaf explants to regeneration and elongation media containing 100 μg·mL-1 concentrations of the antibiotics carbenicillin (crb), cefotaxime (cef), and cefoxitin [=mefoxin (mef)], singly or in combination for 52 days did not eliminate A. tumefaciens from the explants. The percentage of regeneration on crb, cef, and mef was 97%, 11%, and 50%, respectively, compared to 67% for the controls. Short-term (1- to 18-hour) vacuum infiltration with 500 μg·mL-1 of any of the above antibiotics did not inhibit regeneration and failed to eliminate A. tumefaciens from leaf explants. Cef (2000 μg·mL-1) did not inhibit the percentage of regeneration and was more effective than crb or mef in preventing growth of A. tumefaciens when vacuum infiltrated into apple leaf explants for 30 minutes. Further experiments demonstrated that the incidence of A. tumefaciens contamination could be reduced to 28% without negatively impacting shoot regeneration by using a 1-hour vacuum infiltration with an acidified medium, an 18-hour vacuum infiltration with cef (5000 μg·mL-1), and a 52-day incubation on regeneration and elongation media containing 100 μg·mL-1 each of mef and crb. Kan resistant, GUS (β-glucuronidase) positive, putative transformants without A. tumefaciens were generated by adding kan (10 μg·mL-1) to the regeneration and elongation media.