Tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants were transformed with a gene for choline oxidase (codA) from Arthrobacter globiformis. The gene product (CODA) was targeted to the chloroplasts (Chl-codA), cytosol (Cyt-codA) or both compartments simultaneously (ChlCyt-codA). These three transgenic plant types accumulated different amounts and proportions of glycinebetaine (GB) in their chloroplasts and cytosol. Targeting CODA to either the cytosol or both compartments simultaneously increased total GB content by five-to sixfold over that measured from the chloroplast targeted lines. Accumulation of GB in codA transgenic plants was tissue dependent, with the highest levels being recorded in reproductive organs. Despite accumulating, the lowest amounts of GB, Chl-codA plants exhibited equal or higher degrees of enhanced tolerance to various abiotic stresses. This suggests that chloroplastic GB is more effective than cytosolic GB in protecting plant cells against chilling, high salt and oxidative stresses. Chloroplastic GB levels were positively correlated with the degree of oxidative stress tolerance conferred, whereas cytosolic GB showed no such a correlation. Thus, an increase in total GB content does not necessarily lead to enhanced stress tolerance, but additional accumulation of chloroplastic GB is likely to further raise the level of stress tolerance beyond what we have observed.

Upon exposure to 20C, the leaves and crowns of rye (Secak cereal L.cv 'Puma') and wheat (Triticum aestipum L. cv 'Norstar' and 'Cappelle') increased in cold hardiness, whereas little change in root cold hardiness was observed. Both root and shoot growth were severely reduced in coldhardened Norstar wheat plants frozen to -11°C or lower and transplanted to soil. In contrast, shoot growth of plants grown in a nutrient agar medium and subjected to the same hardening and freezing conditions was not affected by freezing temperatures of -20C while root growth was reduced at -15'C. Thus, it was apparent that lack of root development limited the ability of plants to survive freezing under natural conditions. Generally, the temperatures at which 50% of the plants were killed as determined by the conductivity method were lower than those obtained by regrowth. A simple explanation for this difference is that the majority of cells in the crown are still alive while a small portion of the cells which are critical for regrowth are injured or killed.Suspension cultures of Norstar wheat grown in B-5 liquid medium supplemented with 3 milligrams per liter of 2,4-dichlorophenoxyacetic acid could be cold hardened to the same levels as soil growth plants. These cultures produce roots when transferred to the same growth medium supplemented with a low rate of 2,4-dichlorophenoxyacetic acid (<1 milligram per liter). When frozen to -150C regrowth of cultures was 50% of the control, whereas the percentage of calli with root development was reduced 50% in cultures frozen to -110C. These results suggest that freezing affects root morphogenesis rather than just killing the cells responsible for root regeneration.Evaluation of the cold hardiness of roots during the winter is difficult due to the frozen soil and the lack of reliable methods for assessing freezing damage. However, it is generally accepted that roots and other protected parts are less cold hardy than the aerial parts of the plant (5, 6, 9-11).In woody species, it has been shown that cold hardening of roots is determined by genotype, soil temperature, and moisture (11 were sown in 15-cm pots in vermiculite and maintained in a growth chamber at 20/15'C, day/night temperature with 14-h photoperiod for 3 to 4 weeks. These plants were supplied with half-strength Hoagland solution weekly. At the three-to fourleaf stage, the plants were transferred to a 20C constant temperature with a 12-h photoperiod for cold hardening up to 2 weeks. A combination of cool white fluorescent and incandescent lighting (9:1) was used, and irradiance in the 400 to 700 nm range averaged 100 w m-2 as determined with a Li-Cor, Li-170 photometer. Harvested plants were thoroughly rinsed with distilled H20 and separated into leaves, crowns, and roots. Leaves and roots were prepared as 1-cm sections from a composite of 60 plants/cultivar. Crowns were cut approximately 2 mm below and 8 mm above the apex for measuring ion leakage, or 5 cm above the apex for regrowth. Samples were wrapped in wet tissue for ...

limheezing of deep uercooled water In cold-hardened 3-year-old stems of 16 woody taxa was studied in mid-January by dfe al termal analysis lme ndation erare and the size of the low Wemperature exodthn (LTE) (Lonicera X), box elder (Acer negundo L.), May day tree (Prunus padus commutata Dipp.), and red osier dogwood (Cornus stolonifera Michx.). The temperature at the time ofcollection was -25°C and the samples were held at -18°C or lower unless otherwise noted. Some samples were stored outside in sealed plastic bags for approximately 2 weeks. The temperature during this period never exceeded -18°C.Hardinss Determinations Hardiness was determined for thawed and nonthawed samples:Thawed Samples. Stem pieces were brought into the laboratory at 21°C and prepared for freezing. Preparation time was approximately 30 min. Stem pieces 8 to 10 cm in length were placed in aluminum weighing dishes, covered with snow and transferred to the freezing chamber at -3°C. Six stem pieces were used for each selected test temperature (10 in total) at 50C intervals. The samples were maintained at -30C for 10 h and then cooled at 2°C/h as described by Gusta et al. (5).Nonthawed Samples. The samples were prepared outside at -200C and transferred to the freezing chamber at -200C. Upon reaching a selected test temperature, the samples were removed from the freezer, thawed at 0°C overnight and then transferred to a sand bed enclosed in plastic. After 2 weeks, the stem pieces were rated for survival by examination of tissue browning.