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Proline is an effective cryoprotectant for the storage of cultured cells of Zea mays L. in liquid N2. Increased freeze tolerance can be achieved by pregrowth for 3 to 4 days in medium containing proline. Cells cryoprotected with proline have an increased recovery potential when compared with cells cryoprotected with dimethylsulfoxide and glycerol. They also show a reduced postthaw viability loss and greater tolerance of a range of postthaw culture conditions. It is suggested that the mechanism of action of proline may be similar to that in its putative role of conferring protection against natural stresses. It may be protecting the cell against solution effects caused by dehydration during freezing. These findings are discussed in relation to other freeze tolerance enhancing treatments.Freeze preservation in LN3 as a means of genome storage can now be applied to a very limited range of plant tissue cultures. The reasons for the present limitations are not fully understood (1,20,24).In biological systems, the two major sources of cryoinjury are ice damage and solution effects, the latter caused by the excessive concentration of intracellular solutes (9, 10). Protection against cryoinjury may be achieved by either freezing slowly to induce dehydration by extracellular freezing, thereby minimizing intracellular ice formation, or alternatively by freezing and thawing very rapidly to prevent dehydration while maintaining a small, innocuous, ice crystal size. The addition of cryoprotective compounds may reduce cryoinjury in either rapid or slow freezing. In the majority of freeze preservation protocols applied to plant cells, cryoprotectants (usually DMSO and/or glycerol) are used in combination with freezing at a slow rate (1, 24). The high water content of plant cells results in an enhanced susceptibility to cryoinjury. At the level of dehydration required to avoid solution effect stresses, it is still normally necessary to thaw very rapidly to minimize ice recrystallization damage (1,20,24).Recovery of cultures after thawing is dependent upon both a high postthaw viability and further survival through a recovery period preceding renewal of normal metabolic functions and cell division (15,20 In the present study, proline has been used as both a cryoprotectant and pregrowth additive (23) for the freeze preservation of cultured cells of maize. It will be demonstrated that this compound is highly effective in both modes of use and is superior in a number of respects to conventional cryoprotectants. MATERIALS AND METHODSA suspension culture of Zea mays L. (a cell line derived from cultivar B73 [inbred] and kindly supplied by Dr. Ingo Potrykus) was maintained in exponential growth under the conditions described by Potrykus et aL (13). Cells were harvested for freezing 3 or 4 days after subculturing. Additionally, a cell suspension was pregrown for 3 or 4 days in medium supplemented with 5 or 10%1o (w/v) L-proline. Cryoprotectants were prepared using analytical grades of L-proline, glycerol and DMSO, at double th...

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A mutant of Nicotiana plumbaginifolia, CKR1, isolated on the basis of its enhanced resistance to cytokinins was found to have a greater tendency to wilt than the wild type (Blonstein et al., 1991, Planta 183, 244-250). Further characterisation has shown that the wiltiness in the mutant is not caused by an insensitivity to abscisic acid (ABA) because the external application of ABA leads to stomatal closure and phenotypic reversion. The basal ABA level in the mutant is < 20% of that in the wild type. Following stress, the ABA level in wild-type leaves increases by approx 9-to 10-fold while the mutant shows only a slight increase. This deficiency in ABA is unlikely to be the consequence of accelerated catabolism as the levels of two major metabolites of ABA, phaseic and dihydrophaseic acid, are also much reduced in the mutant. The qualitative and quantitative distributions of carotenoids, the presumed presursors of ABA, are the same for the leaves of both wild type and mutant. Biosynthesis of ABA at the C15 level was investigated by feeding xanthoxin (Xan) to detached leaves. Wild-type leaves convert between 9-19% of applied Xan to ABA while the mutant converts less than 1%. The basal level of trans-ABA-alcohol (t-ABA-alc) is 3-to 10-fold greater in the mutant and increases by a further 2.5-to 6.0-fold after stress. This indicates that the lesion in the wilty mutant of N. plumbaginifolia affects the conversion of ABA-aldehyde to ABA, as in the flacca and sitiens mutants of tomato and the droopy mutant of potato (Taylor et al., 1988, Plant Cell Environ. 11, 739-745; Duckham et al., 1989, J. Exp. Bot. 217, 901-905). Wild-type tomato and N. plumbaginifolia leaves can convert trans-Xan into t-ABA-alc, and Xan into ABA, while those of flacca and the wilty N. plumbaginifolia mutant convert both Xan and t-Xan to t-ABA-alc.

STREET. 1973. Control of growth and cell division in plant cell suspension cultures. Can. J. Bot. 51: 1807-1823. In batch suspension cult~lres variation occurs in the growth and metabolism of the cells both in space and with time. Viable cell pop~~lations of sycamore (Acer pselrdol~lata~~lrs L.), showing greatly reduced aggregation and more uniform mor hology, can be obtained by incorporating enzymes into the culture medium. Such techniques cornbinefwith single cell cloning will take us closer to uniformity within the culture. The problem set by the continuous change in the metabolic activities of the cells with time (during the progress of the growth cycle of batch cultures) has been overcome by the use of low-density synchronous cultures and by the establishment of steady states of growth in chemostat cultures. Experimental work with sycalnore cell suspensions is described showing (1) the achievement of prolonged cell division synchrony in 4-liter suspension cultures and (2) the conformity of the growth kinetics of cells in chemostat culture to the mathematical model developed by Monod (1950, Ann. Inst. Pasteur (Paris), 79: 39&410) for microorganisnls. KING, P. J., K. J. MANSFIELD et H. E. STREET. 1973. Control of growth and cell division in plant cell suspensioil cultures. Can. J. Bot. 51: 1807-1823. Lors u'on cultive des suspensions de cellules en lot, des variations se produisent dans la croissance et le mtta%olisme des cellules en fonction de la situation spatiale et du temps. Des populations de cellules viables de sycomore (Acer pse~l~iaplam~lus L.) montrant une tendance B I'agrCgation considCrablement rCduite et une morphologie plus uniforme, peuvent Ctre obtenues en incorporant des enzymes dans le milieu de culture. De telles techniques, associkes B celles du clBnage de cellules isolCes, nous permettrons d'obtenir de plus en plus d'uniformitC dans les cultures. Le probEme, dCcoulant du changement constant des activitCs mCtaboliques des cellules en fonction du temps, (pendant le dkroulement du cycle de croissance dans les cultures en lot), a CtC surmontk par I'utilisation de cultures synchrones i faible densit6 cellulaire, et par le maintien d'un taux de croissance constant en culture en chemostat. Le travail expCrimental rCalisC sur les suspensions de cellules de sycomore est decrit; il montre qu'une synchronic prolongke des divisions cellulaires a Ct C obtenue sur des cultures de 4 litres de suspensions de cellules, et que la cinitique de la croissance des cellules cultivCes en chernostat obeit au modkle mathCmatique dCcouvert par Monod (1950, Ann. Inst. Pasteur (Paris), 79: 39@410), pour les microorganismes. Introduction(as growth in batch culture proceeds). Efforts This paper will examine two propos~t~ons ill have therefore been directed towards the dethe light of currellt research achievemellt from velopment of free-cell cultures and to the estabour own other laboratories. These proposi-lishment of cultural conditions leading to steady tions are (i) that plant cell suspension cultures states of grow...

Selection for cytokinin resistance by incubating M2 seed of Nicotiana plumbaginifolia, after ethylmethanesulphonate mutagenesis, on 20 μM 6-benzylami-nopurine resulted in the isolation of a monogenic, recessive mutant, CKR1. Germination of the mutant is less sensitive to cytokinin inhibition than the wild type, and leaf development of the mutant occurs at cytokinin concentrations inhibitory to the wild type. Germination of the mutant is also resistant to auxin but not to abscisic acid. Three other traits jointly inherited with cytokinin resistance in the F2 are lack of root branching, precocious germination and wiltiness. The wilty phenotype is the consequence of the failure of stomatal closure during water stress.

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Cell division was synchronised in 4-litre batch cultures of Acer pseudoplatanus L. by starvation and regrowth. Up to five consecutive cell cycles were observed in each culture. Mitosis and cytokinesis were synchronised within 0.2 cell cycles. Accumulation of extractable DNA was discontinuous and separate from cytokinesis. Correction for the degree of synchrony in the population gave: G1=13-37 h, S=15 h, G2=14-19 h and M=0.9-1.3 h. Thymidine kinase activity and [(14)C]thymidine incorporation were highest during S-phase. A peak of activity of aspartate transcarbamoylase occurred during G2. Peaks in succinate dehydrogenase activity and respiration rate were observed at the initiation of DNA synthesis and just prior to mitosis. The activity of glucose-6-phosphate dehydrogenase doubled in one step during the cell cycle. Total RNA and protein accumulated continuously through the cell cycle; the final rate being twice that observed initially.