Effect of Chilling Temperatures upon Cell Cultures of Tomato

Abstract

ABSTRACr MATERIALS AND METHODSThe effect of chilling temperatres upon cell cultures of tomato (Lycopersicon eaculektum Mill cv 'VF36,' and cv 'VFNT Cherry,' and L hirsutam Humb. & Bonpl.) was tested. Doubling times for L escuketum were 2 to 3 days at 28°C, and 3 to 8 days at 12°C. No (12,14). L. hirsutum is reported to show greater tolerance to chilling temperatures than the domestic tomato (3, 7, 12-15, 18, 20 (11), 100 LM iron EDTA, 3% sucrose, 2 mg/l 2,4-D, and 0.1 mg/l BA or isopentenyl adenine. The medium is modified from the procedures of S. E. Barsel (personal communication). Cultures were maintained on orbital shakers at 180 to 200 rpm or on a reciprocating shaker at 64 cycles min-'. All stock cultures were maintained at 28C. 'VF36' and L. hirsutum stocks were maintained in 200 ml of medium in 500 ml flasks and transferred every 7 to 10 d by transferring 25 ml of cells to 175 ml of fresh medium. Stock cultures of 'VFNT Cherry' were maintained in 125 ml flasks.Callus from all tissues was initiated on NH4NO3 medium. Suspension cultures were initiated from 2 to 6 months after initiation of callus, and used for experiments from 2 months to 1 year afterwards. Suspension cultures of L. esculentum cv 'VENT Cherry' were maintained in NH4N03 medium, while cultures of cv VF36 and L. hirsutum were maintained in ammonium medium. The cultures grew well in either medium, results were similar using either medium, and choice ofNH4NO3 or ammonium media was not significant to the behavior of the cultures.Growth Measurements. Several methods were used to assess the effect of temperature on growth.A. Weight Determinations. Stock cultures were maintained in 200 ml of medium in 500 ml flasks at 28C. Inoculum from a culture in the early stationary phase of growth was diluted 1 to 5 into 30 ml of medium in a series of 125 ml flasks. The flasks were immediately transferred to the indicated temperatures. At 2 d intervals, flasks were harvested by vacuum filtration onto Whatman No. 4 filter paper on a Buchner funnel, and fresh weight and dry weight were determined.B. SCM3 Determinations Using Stationary Phase Cells. A nondestructive means ofmeasuring the growth ofthe cell cultures was needed. The cultures were thick; also the cells settled rapidly when not agitated, so measurements of the optical density of the cells in a side arm did not seem appropriate. Instead, a method was developed to measure the settled cell volume. Inoculum from a culture in the early stationary phase ofgrowth was diluted 1 to 9 into 45 ml ofmedium in a series of 300 ml side arm flasks, 3Abbreviation: SCM, settled-cell-to-medium ratio.

Adaptation and Growth of Tomato Cells on the Herbicide 2,6-Dichlorobenzonitrile Leads to Production of Unique Cell Walls Virtually Lacking a Cellulose-Xyloglucan Network

“…Suspension-cultured cells of tomato (Lycopersicon esculentum VF36) were obtained from B. Williams (Plant Cell Research Institute, Dublin, CA) and grown on MS medium as described by that group (7). Cells were adapted to growth on DCB by stepwise transfers in gradual increments on increasing levels of DCB beginning at 1 ,aM DCB.…”

Chilling Sensitivity of Cucumber Cotyledon Protoplasts and Seedlings

“…Damage was severe in the presence of glucose, but was markedly reduced in the presence of mannitol, suggesting that injury was related to enhanced cell division potential of protoplasts in the glucose medium. Studies by Breidenbach and Waring (1) indicated that tomato cell suspensions and seedlings responded comparably when subjected to chilling stress, while DuPont et al (2) found that chilling temperatures inhibited growth, but did not kill tomato cells in suspension culture.…”

Preparation of Membrane Vesicles Enriched in ATP-Dependent Proton Transport from Suspension Cultures of Tomato Cells

“…Methods were as previously described (6). Callus was initiated from roots (growth curve experiment) or hypocotyls (all other experiments) of aseptic tomato seedlings (Lycopersicon esculentum Mill cv VF36) germinated on agar.…”

“…The cultures were maintained in 200 ml of medium in 500 ml flasks on a reciprocating shaker at 28°C. The medium (6) Preparations enriched in sealed vesicles were prepared by centrifugation onto a cushion of 10% 70 kD Dextran (4,18). Growth Curve Experiments.…”

“…We investigated the effect of chilling temperature on suspension cultures of tomato cells (6) and planned to examine the effects of chilling temperatures on proton transport in membrane vesicles isolated from the suspension cultures (7). Prior to doing such studies, it was necessary to optimize the conditions for obtaining the proton translocating vesicles from tomato cells and to characterize the vesicles and determine their origin.…”