ABSTRACFThe tissue specificity of the heat-shock response is maize was investigated. The ability to synthesize heat shock proteins (hsp) at 40°C, as well as the intensity and duration of that synthesis, was analyzed in coleoptiles, scutella, green and etiolated leaves, snspensin-cultured cells, germinating pollen grains, and primary root section at different stages of development. One-dimensional sodium dodecyl sulfate gel electrophoresis of extracted proteins revealed that most of the tissues synthesized the typical set of 10 hsp, but that the exact charcterstics of the response depended upon the tissue type. While elongating portions of the primary root exhibited a strong heat shock response, the more mature portions showed a reduced ability to synthesize hsp. Leaves Higher plants have been shown to respond to elevated temperatures by synthesizing a set of hsp3 (1,5,11,18 In this report, the heat-shock response was investigated in a variety of maize tissues, including coleoptiles, scutella, green and etiolated leaves, suspension-cultured cells, germinating pollen gains, and primary roots at different stages of maturity. The ability ofthe tissue to synthesize hsp, as well as the intensity and duration of that synthesis, were analyzed. Also presented is evidence that hsp accumulate only to very low levels in maize. MATERIALS AND METHODSPlant Materials and Incubation Conditions. For studies involving roots, coleoptiles, scutella, and leaves, Crow's Hybrid No. 222 corn (Zea mays L., var A632 x C1042) was used. Roots coleoptiles were obtained from 3-d-old seedlings which had been germinated in darkness at 29°C in gla trays on paper towels kept moist with 0.1 mm CaCl2 solution. Scutella were from seeds imbibed for 24 h under similar conditions.Roots were excised and incubated in the manner previously described (11). Coleoptiles were separated from the rest of the plant, the 1-mm tip removed, and the 1 cm segment immediately below used for the experiment. The segment was longitudinally sliced in half, with one-half used as the control and the other half as the treatment. Half-segments from five plants were combined as a single sample and incubated in the major and minor salts of MS (27) medium as previously described (1 1). Scutella were isolated by aseptically removing the embryos from the seed and then dissecting out the embryonic axis. The scutella were incubated in pairs in a flask containing 2 ml MS salts. The flasks were shaken at 120 cycles/min in a constant temperature bath. The medium was replaced by 2 ml fresh MS salts at the appropriate temperature prior to addition of radioactive label. Unless otherwise stated, roots, coleoptiles, and scutella were incubated at 28°C for 4 h to allow them to overcome the shock of excision prior to any additional temperature treatment. All manipulations involving scutella and coleoptiles occurred in dim green light.For experiments involving leaves, seeds were planted in plastic dishpans in vermiculite and germinated in darkness at 29°C. Once the coleoptiles had emerged thro...

CeHl suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) reitance genes. Resistant

Transient expression of electroporated DNA was monitored in protoplasts of several monocot and dicot species by assaying for expression of chimeric chloramphenicol acetyltransferase (CAT) gene constructions. Expression was obtained in the dicot species of Daucus carota, Glycine max and Petunia hybrida and the monocot species of Triticum monococcum, Pennisetum purpureum, Panicum maximum, Saccharum officinarum, and a double cross, trispecific hybrid between Pennisetum purpureum, P. americanum, and P. squamulatum. Recovery and viability of protoplasts after electroporation decreased with increasing voltages and capacitance while CAT activity increased up to a critical combination of voltage and capacitance beyond which the activity dramatically decreased. The optimal compromise between DNA uptake and expression versus cell survival was determined for D. carota and applied successfully to the other species. Maximum transient expression occurred 36 hours after electroporation of D. carota. The potential for using this procedure to rapidly assay gene function in dicot and monocot cells and application of this technique to obtain transformed cereals is discussed.

Suspension cultures of carrot (Daucus carota, line Cl), tobacco (Nicotiana tabacum, line TXI), and Nicotiana plumbaginifolia (line NP) were frozen under controled conditions with trehalose as the sole cryoprotectant. Maximal post-thaw viability (71-74%), measured by phenofni"n dye exclusion, was obtained with the Cl cells following a 24-hour pretreatment with 5 or 10% trehalose and with 40% trehalose as the cryoprotectant during freezing. TXl nisms accumulate during dehydration high concentrations of trehalose, an a-a-linked glucose disaccharide, which is utilized during and after rehydration. In studies with artificial membranes, it appears that trehalose substitutes for water molecules in the membrane during dehydration and thus helps to maintain membrane integrity (5). Because of these observations, and because structural water is an important component of freezeinjury, we tried trehalose as a cryoprotectant. MATERIALS AND METHODSCarrot (Daucus carota L. var sativa, cv Danvers, root-derived, Trehalose (40% in culture medium) and DMSO (both purchased from Sigma Chemical Co.) were filter sterilized and added to the culture medium to final concentrations of 5 or 10%. Pretreatments began on day 4 for the Cl and TX 1 cells and day 2 for NP cells after subculture.The freezing and thawing protocol was as described earlier (7). Cells were collected from 10 ml medium by centrifugation at lOOg for 10 min. The supernatant was removed and the cells were resuspended in 5 ml of the cryoprotectant solution. Twoml aliquots of this suspension were then placed in Cryotubes (Neslab) which were placed in ice for 1 h prior to cooling at 1°C min-' to -40C. The tubes were then placed in liquid N2 for 2 d before thawing in a 40°C water bath.Cell viability was measured by counting cells which had excluded phenosafranin (9) (about 150 cells were observed for each treatment). The remaining cells were plated on 20 ml of the culture medium solidified with 0.8% Bacto agar in 10-cm plastic Petri dishes which were then incubated at 27 to 28C. RESULTS AND DISCUSSIONInitial experiments using trehalose as the sole cryoprotectant during the freezing period without pretreatment did not allow the recovery of any viable cells following thawing with lines C1, TX 1, and NP. However, the pretreatment of Cl cells with 5 or 10% trehalose for 24 h prior to freezing in 20 or 40% trehalose gave more than 50% viability (Table I; Fig. IA). The cells were capable of continued growth when plated on agar-solidified medium (Fig. IA). The use of 5% DMSO plus 10 or 20% trehalose did not increase the post-thaw viability and did prevent further cell growth in the case of 5% DMSO plus 20% trehalose (Table I).

Five clones were isolated from five different amino acid analog-resistant Daucus carota L. var. Sativa and Nicotiana tabacum L. cv. Xanthi cell lines. The individual clones were similar in their resistance to DL-5-methyltryptophan, S-(2-aminoethyl)-L-cysteine, or azetidine-2-carboxylic acid, and in their corresponding free amino acid levels.The cell suspensions were stored using a controlled freezing rate at -196°C with concentrations up to 40% of the four cryoprotectants: mannitol, proline, dimethylsulfoxide, glycerol, and combinations of dimethylsulfoxide and glycerol. No less than 55% post-thaw viability, determined by phenosafranin dye exclusion, was obtained after storage using a cryoprotectant mixture of 10% glycerol and 10% dimethylsulfoxide. Growth of the cryostored cells could be obtained consistently only by using feeder plate methodology with this combination of cryoprotectants. Post-thaw viabllity and percentage of cells demonstrating growth, as estimated by growth kinetics, were found to be similar. This indicates that little selection occurred during the freezing and recovery process. In addition, the amino acid analog-resistant traits were unaltered following cryostorage.Suspension cultures of Datura innoxia Mill. were frozen similarly with maximum post-thaw viability of 38%, but subsequent growth was not obtained.Protoplasts of D. innoxia, tobacco and carrot were also cryostored using a mixture of 10% dimethylsulfoxide and 10% glycerol as cryoprotectants. Viabilities of no less than 40% were obtained, however, only the carrot protoplasts regenerated cell walls and underwent cell division.The selection of variants through plant cell culture has provided invaluable material to study the biochemical, physiological, and genetic nature of plants. Unfortunately, the maintenance of these variants and wild type lines through serial subculture, either in callus or suspension, is not only expensive, but a number of undesirable genetic changes may occur with time, such as increased ploidy (7), accumulation of spontaneous mutations, and loss of morphogenic competency (6).Cryostorage in liquid N2 may alleviate these problems, but questions concerning its application for genome preservation have been raised. There is concern that there may be selection for unrepresentative cell types which can preferentially survive the freezing and recovery process, particularly in cases where only a small proportion of the cell population may demonstrate regrowth (6). Whether the freezing and thawing processes cause permanent changes in the cells is also a valid question. '