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- Author or Editor: F. Hammerschlag x
Immature `Redhaven' peach (Prunus persica L. Batsch) embryos were infected with Agrobacterium tumefaciens strain tms328::Tn5 carrying the functional cytokinin gene. Shoots were regenerated from callus grown on MS medium without added phytohormones and subsequently rooted on half-strength MS medium with 2.8 -naphthaleneacetic acid. These plants exhibited an increased frequency of branching in vitro. Low levels of cytokinin gene transcripts were detected in these cells by Northern analysis, and using an ELISA assay, the cytokinins zeatin and zeatinriboside were determined to be on the average 30-fold higher. From these results, the expression of the cytokinin gene appears to promote growth of cells in the absence of phytohormones thus serving as a marker for transformation and a promoter of morphogenesis without a 2,4-dichlorophenoxyacetic acid inductive step.
As part of a program to develop transgenic highbush blueberry (Vaccinium corymbosum L.) cultivars, studies were conducted to determine optimum conditions for high-efficiency shoot regeneration from leaf explants of in vitro propagated, commercially important, tissue culture-recalcitrant `Bluecrop' shoot cultures. The effects of pretreatments, growth regulators, and age of explant source on shoot organogenesis were investigated. A maximum of 98% shoot regeneration and 10 shoots regenerating per leaf explant occurred when explants of 2-week-old shoot cultures were incubated in the dark (for a total of 14 days) on pretreatment medium #1 containing 2.6 μM NAA and 5 μM TDZ for 4 days, next on pretreatment medium #2 containing 2.6 μM NAA and 7 μM zeatin riboside for 3 days, then on regeneration medium containing 1 μM TDZ for 6 weeks, and last on medium without growth regulators for 10 days. No shoot regeneration occurred if explants were incubated without exposure to pretreatments before incubation on regeneration medium. There were no significant differences in percentage of regeneration or the number of shoots regenerating per explant from leaf explants derived from either 1-, 2-, or 3-week-old shoot cultures. Shoot production per explant on 1 μM TDZ was about three times that on either 0.5 μM TDZ or 20 μM zeatin riboside, and nine times that on 5 μM TDZ.
A detached-leaf bioassay was used to evaluate peach [Prunus persica (L.) Batsch] somaclone 122-1 (derived from callus produced on an immature embryo of peach cultivar Redhaven) for resistance to several virulent strains of Xanthomonas campestris pv. pruni [E.F. Sm.) Dows], causal agent of bacterial leaf spot, and to a virulent isolate of Pseudomonas syringae van Hall pv. syringae, causal agent of bacterial canker. The detached-leaf bioassay was also used to evaluate progeny of 122-1 for resistance to X. campestris pv. pruni virulent strain XP1. Somaclone 122-1 was significantly more resistant to most strains of X. campestris pv. pruni than was `Redhaven', and all of its progeny exhibited high levels of resistance to X. campestris pv. pruni strain XP1. Somaclone 122-1 exhibited significantly higher levels of resistance to Pseudomonas syringae pv. syringae than did `Redhaven' and this resistance was retained over time in the greenhouse and following a 2-year cycle of tissue culture propagation.
As part of a program to develop transgenic highbush blueberry (Vaccinium corymbosum L.) and strawberry (Fragaria ×ananassa Duchesne) cultivars with increased levels of disease resistance, we have investigated the feasibility of introducing genes for the antimicrobial peptides cecropin B and MB39, α-thionin DB4 (DB4) and γ-thionin RsAFP1 (RsAFP1) by testing the effects of these peptides on several important pathogens of these two crop species. A thin-layer plate bioassay was conducted with these peptides and the pathogens Botrytis cinerea (Pers.ex. Fr.), Botryosphaeria dothidea (Mouq.ex. Fr.) Ces & de Not., Colletotrichum acutatum Simmonds, C. gloeosporioides (Penz.) Penz.et Sacc., C. fragariae Brooks, Monilinia vaccinii-corymbosi Reade (Honey), Phytophthora fragariae Hickman and Xanthomonas fragariae Kennedy and King. The minimum lethal concentration (μm) for cecropin ranged from 0.02 for X. fragariae strains 10 and 128 to 72.8 for C. gloeosporioides isolate Akp1. For DB4, the minimum inhibitory concentration (μm) ranged from 0.03 for X. fragariae strain 6 to 87.2 for B. cinerea isolate cc. For RsAFP1, the minimum inhibitory concentration (μm) ranged from 0.13 for X. fragariae strain 6 to 61.4 for M. vaccinii-corymbosi isolate 9423-x-45. These results indicate that introducing genes for either cecropin, DB4 or RsAFP1 into strawberry may be useful for controlling bacterial angular leaf spot disease caused by X. fragariae.
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.
A wick bioassay was developed to screen peach [Prunus persica (L.) Batsch] shoot cultures for resistance to bacterial spot [Xanthomonas campestris pv. pruni (E. F. Sm.) Dows)]. Three weeks after inoculation, the number of colony forming units (CFU) from 1-cm stem sections excised from highly susceptible cultivars was significantly greater than CFU from several resistant cultivars. Neither growth regulators nor the length of time that shoots were maintained in vitro prior to inoculation significantly influenced the response of these cultivars to bacterial leaf spot. This technique should be useful in screening for somaclonal variants obtained from peach cell cultures.
Shoot tips (5 mm) of Myrobalan plum were cultured on Stage I micropropagation medium consisting of liquid modified Murashige and Skoog salts (MS) medium supplemented with 2% sucrose and in mg/liter: 0.4 thiamine HCl, 100 myo-inositol, 0.5 pyridoxine * HCl, 0.5 nicotinic acid, 0.1 p-aminobenzoic acid, 0.01 indolebutyric acid (IBA) and 0.26-benzylamino purine (BA). A 10-fold multiplication rate was achieved every 4–6 weeks when cultured shoots were transferred to Stage II multiplication medium consisting of Stage I medium with 1.0 mg/liter BA and 0.6% agar. Complete rooting (100%) occurred at 4 weeks when in vitro-proliferated shoots were transferred to MS medium supplemented with 2.5 – 5.0 mg/liter indoleacetic acid (IAA) and incubated in the dark for 2 weeks at either 21° or 26°C. Poor rooting occurred in the light (1.0 klx, 16-hr photoperiod). Shoots incubated at 26°C rooted more quickly than those at 21°C. Shoots cultured on MS medium with IAA and incubated in the dark for 2 weeks rooted significantly better than shoots on medium with IBA and incubated under dark or light conditions. Addition of chlorogenic acid to the rooting medium significantly increased rooting in the light. Plantlets were successfully transferred to soil.
Contamination was reduced from 100 to 3.3% if surface-sterilized forced lateral shoots of peach (Prunuspersica (L.) Batsch), dissected to 0.5–1.0 cm, were cultured rather than similarly sterilized dormant lateral buds which had met their chilling requirement. Optimum shoot tip growth was obtained on a liquid Murashige and Skoog salts medium supplemented with 0.01 mg/liter indolebutyric acid (IBA) and 0.2 mg/liter 6-benzylamino purine (BA). Growth on liquid media was significantly greater than on solid media. Survival of shoot tips at 21° – 24°C was significantly greater than at 26° or 30°. Peach cultivars differed significantly in their survival in vitro.
Callus was induced from anthers of peach [Prunus persica (L.) Batsch] at several stages of pollen development. Cold pretreatments of isolated anthers did not enhance callus formation. Maximum callusing of ‘Sunhigh’ and ‘Compact Redhaven’ anthers was 71% and 87%, respectively, which occurred when anthers containing uninucleate microspores (35 μm) were floated on shallow layers of liquid medium and transferred to fresh medium after 7, 10, and 14 days. Cytofluorometric measurements (UV excitation) of 4, 6-diamidino-2-phenylindole (DAPI)-stained nuclei suggested that the majority of cells were haploid.
Explants from haploid, diploid, and tetraploid geraniums (Pelargonium ⨯ hortorum Bailey) were grown under 1, 5, and 10 klx of continuous cool white fluorescent lamps. Maximum callus production, measured by fresh weight increase occurred at 5 klx for ‘Kleine Liebling (haploid) and at 1 klx for ‘Sprinter’ (diploid) and ‘Sincerity’ (tetraploid). All cultivars grew poorly at 10 klx. Increase in fresh weight of ‘Sprinter’ tissue was 3 times greater than that of ‘Kleine Liebling’ and ‘Sincereity’ at 1 klx and 2 times greater at 5 klx. Differences among light treatments and among cultivars were more apparent with explants taken in winter versus explants taken in the summer.