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László Szalay, Béla Timon, Szilvia Németh, János Papp, and Magdolna Tóth

developed in several stages ( Howell and Weiser, 1970 ; Tromp, 2005 ). Studies on the frost tolerance of the overwintering organs of woody plants demonstrated that the hardening processes taking place during the first half of dormancy could be divided into

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Barbara M. Reed

Cold hardening is an effective method for conditioning meristems for cryopreservation. ABA plays a role in hardening and produces increased hardiness in suspension cultured cells. This study was designed to determine if growth, in vitro, on ABA (5×10-5 M) for one week, would substitute for one week of cold hardening, and if ABA would provide additional conditioning when added in combination with cold hardening treatments. In vitro plantlets of Rubus spp. were grown for one week with or without cold hardening and with or without ABA. Meristems from these plants were frozen at 0.8C* min-1 to -35 C, then plunged into LN2, thawed, and plated on recovery medium. One month after thawing, cold-hardened plants with and without ABA treatment had recovery rates of up to 83%. Survival of plants grown at room temperature ranged from zero to 8% and zero to 28% for plants grown on ABA at room temperature. At the rates tested, ABA is less effective than cold hardening in conditioning apical meristems of in vitro Rubus plants for cryopreservation and provides no additional protection to cold-hardened meristems.

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Diana L. Dostal and Arthur C. Cameron

Postharvest shelf life of fresh sweet basil (Ocimum basilicum L.) at 5°C is only 3 to 4 d due to development of chilling injury symptoms. Plants chill-hardened at 10°C for 4 h daily (2 h at end of the light period and 2 h at the beginning of the dark period) for 2 d prior to harvest had 3 d extended shelf life at 5°C. Increasing the duration of preharvest chill-hardening did not further improve the shelf life. Plants were chill-hardened at 10°C for 4 h daily for one week at different periods during the day. Four-, 5- and 6-week-old basil were used in each of three consecutive runs. With the 4- and 5-week-old basil, chill-hardening at the beginning of the day extended average shelf life by 1 and 1.5 d at 5°C, respectively. Shelf life was either decreased or not affected by the other periods of preharvest chilling. Postharvest chill-hardening of packaged sweet basil for 1 d at 10°C before transfer to 5°C increased shelf life by 5 d. Postharvest chill-hardening has potential for reducing chilling injury of packaged sweet basil.

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Jennifer Crane and Harrison Hughes

Plantlets of Solarium tuberosum L. `Russet Burbank', `Sangre', and `Centennial Russet' were grown in vitro from nodal cuttings. A medium overlay was used to reduce the humidity of the in vitro environment. This treatment was tested for its effect on plant growth and on the rate of water loss from detached leaves. The latter was assayed as indicative of hardening and consequent survival of plantlets once removed from in vitro culture. The paraffin medium overlay reduced the rate of water loss from detached leaves of cultured plantlets, but also reduced root growth.

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Warren Roberts, Jim Duthie, and Wes Watkins

Wet soils can prevent growers from transplanting tomatoes at the ideal size and age. Experiments were conducted to determine the length of time that transplants can be held before yield is reduced Also, different techniques for holding and hardening plants were compared. Seven ages of `Sunny' tomato plants (4, 5, 6, 7, 8, 9, 10 weeks old at transplanting) were either grown normally, grown with limited water, or grown with limited fertilizer. Plants were grown in trays containing 128 cells, with each cell approximately 3.2 by 3.2 by 11 cm. Water was applied for 3 minutes either once a day or twice a day. Fertilizer (20-20-20) was applied either once a week or once during the entire seedling production period. Transplants were later planted in the field. The experiment was conducted in 1990, 1991, and 1993. The yield response to transplant age was quadratic, with maximum yield occurring with 6, 7, and 8 week old transplants. In general, the greatest yield occurred when water was withheld, and the lowest yield occurred when fertilizer was withheld from the transplants

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George Yelenosky

Potted greenhouse-grown, l-year-old `Hamlin' orange [Citrus sinensis (L.) Osbeck] trees on 1.5-year-old rough lemon (C. jambhiri Lush.) rootstock were temperature-conditioned for 6 consecutive weeks in a controlled-environment room to test cold-hardening ability. Holding at 15.6 ± 0.6C during 12-hr days [425 μmol·s-1·m-2 photosynthetic photon flux (PPF) at top of trees] and 4.4C during nights resulted in 100% tree survival and no leaf loss “after 4 hr of – 6.7C in a dark freeze test room. Unhardened greenhouse trees were killed to rootstock. Solute efflux (dS·m-1) from unhardened frozen leaves was > 20-fold that from frozen leaves on hardened trees and nonfrozen leaves on unhardened trees. Oxygen uptake was not significantly impaired in frozen hardened leaves. No 02 uptake was evident for frozen unhardened leaves.

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Ribo Deng and Danielle J. Donnelly

Micropropagated `Festival' red raspberry (Rubus idaeus L.) shoots were rooted in specially constructed plexiglass chambers in ambient (340 ± 20 ppm) or enriched (1500 ±50 ppm) CO2 conditions on a medium containing 0, 10, 20, or 30 g sucrose/liter. Plantlet growth and leaf 14CO2 fixation rates were evaluated before and 4 weeks after ex vitro transplantation. In vitro CO2 enrichment promoted in vitro hardening; it increased root count and length, plantlet fresh weight, and photosynthetic capacity but did not affect other variables such as plantlet height, dry weight, or leaf count and area. No residual effects of in vitro CO2 enrichment were observed on 4-week-old transplants. Sucrose in the medium promoted plantlet growth but depressed photosynthesis and reduced in vitro hardening. Photoautotrophic plantlets were obtained on sucrose-free rooting medium under ambient and enriched CO2 conditions and they performed better ex vitro than mixotrophi plantlets grown with sucrose. Root hairs were more abundant and longer on root tips of photoautotrophic plantlets than on mixotrophic plantlets. The maximum CO2 uptake rate of plantlet leaves was 52% that of greenhouse control plant leaves. This did not change in the persistent leaves up to 4 weeks after ex vitro transplantation. The photosynthetic ability of persistent and new leaves of 4-week-old ex vitro transplants related neither to in vitro CO2 nor medium sucrose concentration. Consecutive new leaves of transplants took up more CO2 than persistent leaves. The third new leaf of transplants had photosynthetic rates up to 90% that of greenhouse control plant leaves. These results indicate that in vitro CO2 enrichment was beneficial to in vitro hardening and that sucrose may be reduced substantially or eliminated from red raspberry rooting medium when CO2 enrichment is used.

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Aaron J. Brown

Polyethylene glycol (PEG) was evaluated for its influence on hardening of in vitro-propagated `Fern' strawberries (Fragaria ×ananassa) when applied just before transplanting. Strawberries were micropropagated via shoot tips and grown in vitro until roots were well developed. Plantlets were then transferred onto filter paper bridges in liquid medium with 15% (w/v) of PEG-8000. After treatment in the medium for various periods, the plants were compared to the control (no PEG) for water loss from detached leaves, stomatal aperture, and survival rates after transplanting. Leaf epicuticular wax was also quantified. Overall, the in vitro PEG treatment was not successful in significantly increasing hardiness and survivability of the strawberry plants after transplanting from in vitro conditions to a soil medium. Osmotic stress was created, but apparently not for the time needed to increase survival. Further tests are needed to pinpoint the proper exposure time required to increase hardiness and survivability after transplanting plantlets. To increase survival, the time exposed to PEG should be 15, 18, or possibly 21 days.

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Kenneth L. Giles, Kenneth Friesen, and Debra Novakovski

The use of an in vitro mist culture systems for the growth of virus free shoots of several potato cultivars has been demonstrated using the “Mystifier®”, several different media and growth regulator concentrations have been used to monitor the efficiency of shoot multiplication and growth. Nodal cuttings of sterile in vitro virus free cultures were used as inocula and the mist was applied at various rates in order to assess optimum sucrose concentrations and mineral concentrations. Murashige and Skoog medium was used and diluted to half and quarter strength.

Hardening off the resulting shoots was achieved by passing HEPA filtered air at various rates over the plantlets still in the culture vessel. Conditions for high efficiency hardening off of cultured material has been defined such that plantlets can be transferred directly to greenhouse conditions.

The implications and opportunities that are indicated by this work are discussed.

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Carme Biel, Robert Savé, Abdessamad Habrouk, Josep Maria Espelta, and Javier Retana

Extensive areas occupied by Pinus nigra forests in Spain have burned in recent years. Recovery of these forests depends upon reforestation. In this study, we analyze the combined effects of different treatments of water restriction and air enrichment with CO2 in the nursery as a means to increase hardening and vigor of P. nigra seedlings of various ages (recently germinated, 1- and 2-year-old) and their post-transplant performance in a burned area. In all cases, 2-year-old seedlings showed the highest specific leaf weight and low cuticular transpiration rates. CO2 enrichment increased total biomass, leaf biomass and leaf area of seedlings while water restriction decreased leaf area, leaf biomass, and stem biomass, without any significant interaction between both experimental factors. Younger (recently germinated and 1-year-old) seedlings showed a larger relative response to the experimental factors than 2-year-old seedlings. After transplanting, the previous CO2 and water treatments did not affect seedling survival, but 2-year-old seedlings showed the lowest mortality rates.