Due to the heterogeneity in morphology, physiological and morphogenetical capabilities of higher plant cells in mass culture, the development of methods for individually culturing defined cells seemed to be useful and necessary. Individual cell culture represents a powerful tool for studies on the physiology of different cell types, the analysis of differentiation programs, the genetic manipulation of plant cells and cell-cell interactions. An improved microculture system based on a computer-controlled set-up for the efficient selection, transfer and individual culture of defined higher plant cells until regeneration of whole plants is described. Related experimental approaches for individually manipulating higher plant cells under controlled conditions, such as electrofusion of defined pairs of protoplasts and subprotoplasts, cell reconstruction and intranuclear microinjection of protoplasts and karyoplasts - mainly performed with cells of the crop plant Brassica napus L. - are presented.
The circadian rhythm in the oxygen production of 30 individual Acetabularia cells has been studied at different temperatures. The temperature induced period variation was continuously evaluated over the whole data record of each individual cell with an advanced spectral analysis technique. The observed circadian periods of O2 production displayed a well established region of temperature compensation between 25 °C and 30 °C with a Q10, value of 0.9, whereas between 15°C and 22°C a positive temperature coefficient was measured (Q10 at 22 °C 0.9, Q10 at 20°C 0.8, Q10at 17°C 0.7).
Two single mesophyll-protoplasts of Nicotiana tabacum cv. xanthi were selected into a 100 nl microdroplet of 0.4 M mannitol. Two cylindrical platinum electrodes were inserted into the microdroplet to align the two single cells via dielectrophoresis in an AC-field (1 MHz, about 120 V X cm-1). A single square DC-pulse of about 1.5 KV X cm-1 was applied to induce protoplast fusion.
A multitasking time-sharing computer system was implemented for studies of different circadian rhythms in individual cells of the unicellular green alga, Acetabularia. This fully automatized system allows simultaneous data acquisition and analysis. Graphical presentation of untreated and mathematically treated data is permanently available on three graphic displays and on a digital plotter. The sampling rate for the data acquisition in each of the 60 channels connected to the system is 720/24 h. Provisions have been made to guarantee uninterrupted data uptake for these long-term measurements by including an auto-restart module and by providing extremely reliable software for the experimenter using menu techniques.
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