S. V. Lebedev and V. P. Chekhov.

Change in the Structure of Sugar-Beet Roots under Freezing.

S. V. Lebedev and V. P. Chekhov.

Changes in the Structure of Sugar-Beet Roots During Freezing

The wide spread of sugar-beet cultivation, both to the West—to North America—and to the East—to Siberia, and the effort to organize a sugar industry there, place before the latter a number of new tasks and questions. These arise both in the field of beet seed-growing and the agriculture connected with it, and in relation to production, i.e., in the sense of the conditions for processing sugar beets. In both cases, in Siberia one must take into account the characteristic features of its climate and, in particular, its sharply expressed continentality. Moreover, for beet seed-growing in Siberia the chief factor in this case proves to be the shortness of the growing season, whereas with respect to factory processing, the greatest importance here should apparently be attached to the great duration of the Siberian winter and to the low temperatures unusual for sugar-producing regions.

Until now, over a long period of years, a stubborn struggle was waged for the very idea of a Siberian sugar industry, concentrated chiefly on the question of the possibility of industrial cultivation of sugar beets in Siberia. Now this question has already been settled, and favorably; but in its place new questions of a different, production-related order are being advanced. Here, above all, attention is drawn to the choice of a method of storing beets suitable for Siberia. It is obvious that under the conditions of a Siberian winter, with temperatures reaching −30, −40°, the ordinary methods of storing beets in covered clamps, with the obligatory concern for maintaining in them a certain temperature above 0°, are little applicable, since episodes of beet freezing in such clamps, undesirable under the conditions of modern European beet-sugar regions, become ordinary and even inevitable for Siberian sugar factories in the coldest months of winter. The meaning and significance of this circumstance is determined by the generally known position that frozen beets, under the conditions of the sugar industry, constitute a deeply damaged raw material, extremely hampering factory operations in general and especially juice extraction. Under such conditions, for the Siberian sugar industry the task arises either of working out new methods of storage with thawing of the beets being stored, or of following the proposal of Prof. Lebedev, put forward by him for the Altai sugar factory as early as 1916—to store beets in the frozen state in open clamps.

This idea proceeded from the following considerations:

1) From the technical and economic difficulty, under Siberian cold conditions, of maintaining millions of poods of beets to be processed by the factory at temperatures above 0°.

2) From the absence in Siberia of winter thaws and, finally,

3) From the fact that the freezing of beets, according to the generally known proposition concerning the connection between respiration, sugar losses, and the temperature of the beets, makes it possible to reduce the sugar losses of the latter to a minimum, while at the same time eliminating various unfavorable changes in the non-sugars of the beets that are inevitable when they are stored at temperatures above zero.

Although the idea just presented—of freezing beets—is logically quite correct for Siberia, nevertheless, in order to put it into practice in the sugar industry, it is necessary to clarify a number of additional questions concerning the possible biological, chemical, structural, and other changes that the sugar-beet root may undergo when stored in a frozen state during the Siberian winter. The significance of these questions is very substantial, both from the standpoint of the tasks of production, which will have to deal with this kind of raw material, and with regard to the storage of the frozen beets themselves—not so much during the winter’s stable frosts as in the period of late winter and early spring, when thaws already become a quite regular phenomenon even in the Siberian climate, and when beets, even if they have not undergone freezing, begin to experience the pressure of both internal and external forces that destroy them. Obviously, the effect of the latter upon beets that have frozen through during the winter must be different from their effect upon fresh beets. Moreover, the effect of the new action will be determined by the degree and character of those changes that were the result of the winter freezing of the beets.

In connection with this, one should expect that, under the conditions of the Siberian winter, the influence of cold on stored beets will differ sharply from what usually takes place in this respect under the conditions of our and Western European old beet-sugar regions, with a climate milder than that of Siberia. This indicates that the frozen beets of future Siberian sugar factories will, in their properties, be an entirely new raw material, unfamiliar to the sugar industry.

It is obvious that the success of factory processing of such raw material will require knowledge of its properties and special features. Thus, the study of the changes undergone by the sugar-beet root when it freezes during the winter period under the conditions of the Siberian climate is one of the essential and responsible tasks in the organization of the Siberian sugar industry; the urgency of this task is determined by the construction of a sugar factory already under way in Altai, and by the fact that until now this question has remained entirely untouched, and the present work is the first step in this direction.

One of the basic questions in the present case lies in determining whether, after freezing, the cells of the tissue of the beet root prove to be alive or dead, and, furthermore, whether they are intact or destroyed. This should be of interest from various points of view: first, with respect to the conditions for storing frozen beets, especially in the spring period; second, with respect to the methods of juice extraction in general and, in particular, with respect to the temperature regime, the rate of work, and other conditions in the factory diffusion battery.

Emphasizing the necessity and importance of studying the properties, structure, and other special features of Siberian frozen beets as the future regular raw material of Siberian sugar factories, it should also be noted that the information available up to now, both from the field of practical factory work and from the literature, is very limited, accidental, and often contradictory. Moreover, all these data do not relate to—

as much to frozen beet as to beet that has already thawed, or that freezes again and then thaws again, and which, because of this, has undergone, in addition to the principal temperature effects, still further complex internal influences of an enzymatic and chemical order, as well as external influences, often considerable, from the microflora.

All these factors, in their combined action upon the beet, produce in it significant biological changes and profound disturbances not only of the chemical, but also of the purely mechanical side, making the mass of the beet root mechanically very weak. As a result, such frozen beet, during factory processing, gives, when sliced, chips that are not very firm and that easily mat together, which, for this reason, greatly hampers the operation of the factory diffusion battery.

In order to illuminate a whole series of questions which, both theoretically and in production, may be of interest to the sugar industry with respect to the properties and peculiarities that the sugar-beet root must acquire after its prolonged freezing under the conditions of a Siberian winter, it would be most expedient to approach this question from the standpoint of elucidating the consequences of these influences upon the basic element of the entire beet root from the point of view of the sugar industry—i.e., upon the cells of its parenchymatous tissue, where the extractable sugar is concentrated. Having established this, it would be easier to orient oneself, to understand, and, to a certain extent, to foresee those changes and consequences of the influence of freezing which, under these conditions, the sugar-beet root as a whole undergoes.

In view of the above, the aim of the present work in what follows is to reveal, by means of microscopic and partly macroscopic investigations, those changes which the tissue of the beet root undergoes under the action of the prolonged cold of a Siberian winter.

The influence of cold on the cells and tissues of plants has been studied by many investigators. In the Russian literature there is, on this question, the very valuable work of Prof. N. A. Maksimov, “On the Freezing and Cold Resistance of Plants.” Prof. N. A. Maksimov and other investigators approached the phenomenon of death from cold from a purely biological point of view. They are interested in the cause of death, and the phenomenon of rupture of the cell membranes is important for them only insofar as it may be the cause of the death of the organism. Despite a different approach and entirely different ultimate aims, these works are also of considerable interest to us.

Investigators of the eighteenth and the beginning of the nineteenth century saw the cause of plant death in the rupture of tissues.

Buffon and Du-Hamel (1737) believed that, during freezing, the sap filling the vessels increases in volume and stretches them. During rapid thawing, when, in the opinion of these investigators, “something like ice drift” begins, the vessels burst, the sap flows out, the supply of water ceases, and the plant dies. Senebier (1800) shares the point of view of the previous authors on the question of the cause of death, but considers that the rupture of the vessels occurs not during thawing, but during freezing, as a consequence of the expansion of the water contained in them. In works beginning with 1830 and up to the present time (Göppert (1830), Sachs (1860), Nägeli (1861), Prillieux (1913)), devoted to the study of the influence of cold, we find that plants die not from the rupture

cell walls, but rather from the more subtle changes undergone by cells under the influence of frost. Göppert (1830) examined under the microscope 222 species of various plants killed by frost and then thawed; in all of them the cell walls remained completely undamaged¹.

The doctrine of the rupture of cell walls disappeared, and the cause of death came to be seen—

1. In the specific action of low temperatures—in the loss by the cell of its “vital force” under the action of frost (the vitalistic point of view—Göppert (1830));

2. in rapid thawing, since the cell membrane and plasma lose water when frozen; the cell sap, turning into ice, becomes more concentrated. If thawing occurs slowly, the water is again absorbed by the cell, and the original properties of the cell sap, membrane, and protoplasm are restored; with rapid thawing, however, part of the water formed flows into the intercellular spaces, and the normal properties of the cell cannot be restored (Sachs (1870)).

Muller-Thurgau (1830, 1886) opposed these two views. This scholar’s experiments with plant leaves, as well as with tubers (potatoes), showed that plants are capable of enduring, without harm, cooling for a long time down to temperatures of −2°, −3°, provided that ice formation does not occur; where ice is formed, the tissues die. Muller-Thurgau, who advanced the theory of dehydration, sees the cause of the death of the organism in the phenomenon of the removal of water by the ice masses formed in the intercellular spaces. Moreover, the first appearance of ice crystals does not always entail death; death occurs only when, for the formation of ice, the cell gives up so much water that the organized structure of the protoplasm is disrupted. Muller-Thurgau’s microscopic investigations led him to the conclusion that in thin preparations subjected to the action of cold, because of rapid cooling, ice forms inside the cells; when entire massive organs of plants freeze, ice forms in the intercellular spaces.

Gorcke (1907) considers that the removal of water during freezing increases the concentration of the cell sap, and the salts present in it salt out protein substances; moreover, prolonged action of a concentrated salt solution can cause irreversible changes in proteins—denaturation. Of greatest interest to us are the works of Molisch (1897), Matruchot and Molliard (1902), and Schaffnit (1910—1911).

Molisch began his microscopic investigations by studying the influence of freezing and subsequent thawing on various colloids, emulsions, and other substances that enter into the composition of the cell. It turned out that after thawing, in some cases the colloid again assumed its original appearance, while in other cases the removal of water occurring during freezing proved to be an irreversible process. Studying plant and animal objects, Molisch fully agrees with the conclusions of Muller-Thurgau.

Phycomyces and the hairs of Tradescantia withstand supercooling down to −9° and remain alive, provided only that they are thawed before ice forms. If, however, supercooling does not occur during freezing, then the organisms perish from the ice that forms at temperatures above −9°. Ice, in Molisch’s opinion, forms during the freezing of plants not only in the intercellular spaces but also inside the cells. Evidence of this may be provided by air bubbles, the presence of which, according to Molisch and Müller-Thurgau, is a sure sign of internal ice formation. Different cells, and still more different tissues, prove to be unequally tolerant of cold; this is explained by the fact that they have different points of possible supercooling. Molisch’s microscopic studies, however, do not give a clear picture of those subtle cytological changes undergone by a cell subjected to the action of cold. This gap was filled to a considerable extent by Matruchot and Molliard, who studied material that had been fixed and then stained. The objects of study were frozen with the aid of a cooling mixture and, in the frozen state, were immersed in the fixative—Flemming’s mixture. The preparations were stained with safranin. As the material for their investigation the authors used the spongy parenchyma of narcissus leaves. The effect of cold was manifested above all in the structure of the nuclei. Normally, fine networks of chromatin were found in the nuclei, within which were nucleoli. In frozen and then thawed specimens, vacuoles are observed in the nuclei; their number is sometimes so considerable that the nucleus appears to consist of separate chambers.

These vacuoles, depending on the position of the nucleus, are arranged bipolarly or at one pole. Sometimes the nucleus turns into a bubble, divided into two parts by a ring of chromatin. In some cases a rupture of the thin layer of the nuclear membrane and protoplasm is observed. The appearance within the nucleus of water-filled chambers is the result of the separation of water from the nuclear substance, which is entirely consistent with Müller-Thurgau’s theory of “dehydration.”

The picture of changes in plant cells subjected to plasmolysis and desiccation is quite analogous to what occurs in the cells of frozen organisms. According to the observations of Matruchot and Molliard, the change in the protoplasm in plant cells that have died from cold consists in the granular or fibrillar structure passing into a foamy one, typical of dead protoplasm.

Müller-Thurgau’s point of view met with severe criticism from Pfeffer (1901), Mez (1905), Apelt (1907), and Rein (1908), who considered that the death of organisms occurs when cooling (even if only for an instant) passes beyond the temperature minimum specific for each organism.

The careful investigations of N. A. Maksimov, who used the thermoelectric method of measuring temperatures, led him to the conclusion that Müller-Thurgau’s conclusions are the most reliable. The teachings of Apelt and Rein, as well as of Görcke, do not withstand experimental verification.

In concluding this review of the literature on the question of freezing injury and cold resistance in plants, it must be noted that the point of view of Müller-Thurgau and Maksimov, although at present the most widespread, still does not allow the question of the causes of the death of organisms during freezing to be regarded as definitively resolved. But,

if, on the question touched upon above, a final conclusion has not yet been reached, there is complete agreement that the membranes of the cells of organisms killed by cold remain intact.

The first question of interest to us was to establish the moment at which the cells of sugar-beet roots die. Whereas in some plants, after death, a rapid change in coloration occurs¹, in sugar beet there are no substances that change their color after the death of the organism; the cell sap is also uncolored, and consequently no change in coloration occurs here at the moment of death. Working with thin sections, it is possible in many cases to determine the condition of the cells on the basis of the phenomenon of osmosis.

But this method, too, proves inapplicable to sugar beet, since the cell sap is uncolored and the amount of plasma is so minimal that it is almost imperceptible even in fixed and subsequently stained preparations; moreover, the cell membranes possess considerable thickness. The circumstances indicated above also preclude the possibility of observing the phenomenon of plasmolysis—the separation of the protoplasm from the membrane. An attempt to apply a staining method with a mixture of dyes (Neutralrot-Methylenblau) was likewise unsuccessful.

Ordinarily, when this method is applied, the plasma of living objects is stained a reddish color, while in dead ones the plasma acquires a blue coloration. When the dye mixture is applied to sugar beet, however, in all cases (in cells known to be living as well as dead) we obtain staining of the membrane in a violet color. The protoplasm itself, which in the present case is the reagent, being present in the cells in negligible quantity and enclosed within thick cell membranes, cannot serve as an indicator of the state of the cells.

Of all the methods we employed under the conditions of our experiment, the suitable ones proved to be macroplasmolysis and the darkening, upon thawing, of sugar-beet tissues killed by frost.

If a section of living tissue of some plant organism is placed in a solution of sugar or salt, we may observe phenomena of two kinds:

If the concentration of the solution surrounding the section is higher than the concentration of the cell sap, then part of the water contained in the cell will pass out (exosmosis), and the protoplasm begins to draw away from the walls—this phenomenon is called plasmolysis. In the opposite case, when the concentration of the cell sap is higher than that of the surrounding solution, water will pass into the cell (endosmosis). The osmotic pressure caused by endosmosis, called turgor, is characteristic, to a greater or lesser degree, of every living cell. In a dead cell the phenomena of exosmosis and endosmosis do not occur. Ludwig Jost, in his lectures on plant physiology (translated from the 2nd German edition, 1912), writes (p. 24): “When de Vries (1877) placed in water thick sections of beet from which all traces of sugar had been carefully washed out of the cells damaged by cutting, he was able to show that even after 14 days sugar had not diffused out of them. If red beet is taken for the same experiment instead of white beet, we find that the protoplasm is impermeable also to the dissolved cell sap

of pigment, as also for cane sugar,” and further on p. 41: “In conclusion we shall also note that endosmosis and exosmosis depend not only on the properties of the plasma, but the cell membrane may also sometimes bring about impermeability for certain substances, especially when it has become corky (Krömer 1903). Since corking after injury may occur within only a few hours (Appel 1906), it is quite possible that the insufficient endosmosis of sugar in de Vries’ experiment with beet (p. 24) depended on such formation of a ‘plug’.” At present it is believed that the phenomena of exosmosis and endosmosis depend almost entirely on the surface, cuticular layer of the protoplasm (Prof. Ivanovsky, Plant Physiology, 1919). If the phenomena of exosmosis and endosmosis are difficult to observe in individual cells when studying thin sections under the microscope, then increasing the volume of pieces of beet (2–4 cm³), owing to the increased osmotic pressure caused by endosmosis, considerably facilitates observation of these phenomena.

For this purpose, pieces of sugar beet of 2–3 cm³ were immersed in distilled water and, on the basis of measurements with an accuracy of up to 0.1 cubic cm, at definite intervals of time it was determined whether their volume increased and, if it did, to what extent.

Thus, the disappearance of the phenomenon of osmotic pressure may be an indicator of the moment of death. Placing slices of fresh beet and frozen beet (which had remained in the open air from 20 October until the experiment—17 January) in distilled water, we found that fresh beet increases considerably in volume (on average by 60%) during the first two hours after immersion in water, while subsequently its volume does not change; beet frozen since autumn, however, preserves its original volume unchanged throughout the whole time.—Slices subjected for 2–3 days to the action of cold increase so insignificantly that in some cases this increase lies within the limits of observational error. If, however, we adhere to the view of de Vries cited by Jost, that the force of osmosis depends on the integrity of the membrane, then we would have to admit that the cells of beet cut into slices and exposed to frost from 15 to 17 January must, to a considerable degree, have undergone rupture, since they showed no phenomenon of osmosis. But careful microscopic investigations convince us of the opposite; therefore it must be thought that it will be more correct to explain the phenomenon of osmosis by adhering to the most recent point of view, namely: osmosis is conditioned by the cuticular surface layer of the plasma, while the cell membrane, if it has any significance, has only a very slight one. The absence of osmosis in the preceding experiments indicates coagulation of the plasma, i.e., the death of the cell, while the cell membranes in this case are preserved.

Taking advantage of the comparatively warm day of 8 January, we carried slices of fresh beet outside, where they remained from 1 p.m. to 9 p.m. During this interval the air temperature fluctuated from −10.8° C at 1 p.m. to −7.8° C at 9 p.m.

After this we immersed them in water and found that their volume had not changed. Consequently, the mean temperature—9.3° C (over 8 hours)—is already lethal for beet in the given case.

In our investigation, the activity of the enzymes that cause darkening of the tissues may also serve as a reliable indicator of cell death.

...cells. The relative resistance of enzymes to the action of cold is a well-known fact. Freezing was used in the works of Palladin (1905), Krasnosel’skaya (1905, 1906), and Kovshov (1906) even as a method for studying the action of intracellular enzymes. In the above-described experiments we carried out, when the tissues had perished from the action of cold, which was confirmed by the absence of the phenomenon of osmosis, the cells, when they were left (owing to the free penetration of air into them and the activity of the enzyme thereby conditioned) took on a dark coloration. The lower the temperature and the longer its action, the more ruptures there were, and the more easily air penetrated into the tissues, the sooner the darkening process proceeded. It must be noted that the action of the enzyme begins already at temperatures close to 0°, and darkening, reaching −2°, causes in roots kept in the open air a darkening that extends far deeper than the region of the vascular bundles, and then to the parenchyma cells. Since in the region of the bundles the vessels are situated closer to one another, rupture here is greater and the surface area of them is more considerable, and therefore darkening occurs here more rapidly.

No less important for us is the study of the structure of the root, affected by the action of cold. The tissues of frozen and fresh beet were studied in sections made with a razor by hand and in preparations prepared with the aid of a microtome. Preparations made with the aid of a razor served in our investigations as a guide for studying the structure of the cells; for studying the structure of cells, a more complicated method of processing the material was used. Usually pieces of fresh beet or beet subjected to the action of cold, 1–2 mm thick, were used and were fixed with the following substances:

1. A mixture of bichromate [[unclear: salt]], for 12–24 hours.

2. A mixture of chromic acid and 10% formalin (in different ratios: 5 : 5, 4 : 5, 7 : 3).

3. Palladin’s mixture: chromic acid 10 cm³, 10% formalin [[unclear: 5 cm³]], glacial acetic acid 1 cm³.

4. Flemming’s mixture (weak concentration): one-percent chromic acid 15 cm³, two-percent osmic acid 2 cm³, glacial acetic acid 1 cm³, distilled water 10 cm³.

Preliminary investigations showed that the best results were obtained when the last two fixatives were used, owing to which the mixture of Palladin and Flemming was usually fixed upon; after fixation, for 24 hours, thin pieces of beet were washed in running water, then treated with alcohol, passed through a mixture of alcohol with xylol, then through pure xylol, and finally placed already in paraffin for 3–4 days in a thermostat (at \(t^\circ\) 50–52° C). Sections were made with the aid of a microtome of Jung, with a thickness of from 6 to 10 microns. Of the various staining methods, the best proved to be staining with hematoxylin by Heidenhain’s method and triple staining with safranin and Blochmann’s mixture¹. The material for investigation consisted of beet roots sent from the Aleisk district, Barnaul okrug, and from the farm of the Aleisk beet-sugar state farm to Tomsk on October 20 (the minimum temperature for the period from 1.X to 20.X was −1.6° C), preserved during the entire time of the experiments under conditions of outside-air temperature in a plank shed, placed in

closed boxes, and fresh beets that had been kept in the laboratory cellar at a temperature of about \(+4^\circ\mathrm{C}\).

The study of the structural changes occurring in beet roots subjected to the action of cold was begun in January 1929. The beets had lain in the above-mentioned rooms and conditions. For a meteorological temperature characterization of the period from October 20 to March 1, we present the following tables, based on data from the meteorological station of the Siberian Technological Institute in Tomsk.

Fluctuation of minimum temperatures by ten-day periods:

Already the first sections at the end of December 1928 revealed clearly visible ruptures—cracks—in the tissues of frozen beet roots, in the form of a straight or broken line. The number of these ruptures was not the same in individual roots. On the basis of our observations it may be concluded that the number and size of the ruptures are directly proportional to the size of the root. The larger the root (the thicker it is), the greater the number of ruptures, which is clearly visible in the photographic images (2–5). Studying our preparations, we may note that where the cut of the microtome knife is perpendicular to the direction of the vascular-fibrous bundles, in all cases it is clearly seen that the vessels remain intact (see photograph 8). Meanwhile, Buffon and Du-Hamel (1737), as well as Senebier (1800), explained the death of the organism by rupture of the vascular-fibrous bundles. From this we may conclude that the vascular-fibrous bundles do not tear even under the conditions of our experiment, when

the beet roots were subjected to extremely severe cooling for a very long time (up to 2–3 months). The rupture occurs in the zone of the interfascicular parenchyma, and the resulting fissure is located midway between the bundles; sometimes, however, especially with more prolonged freezing (over 2–3 months), one or two additional fissures appear on either side of the first, but their dimensions are somewhat smaller.

The fact established here, that cells rupture, does not entirely agree with the currently prevailing and widely accepted view that cell membranes are preserved during freezing. This contradiction compelled us to dwell on this question in greater detail and, in the investigation, to give it special attention.—In addition to mutual checking of sections made by hand with a razor and with the aid of a microtome, we took into account and eliminated possible causes which, during preparation of the specimens, might have produced such ruptures. The position of the ruptures always remained the same, regardless of how the vascular-fibrous bundles were oriented—along the length of the paraffin ribbon or across it. The observation of the rupture phenomenon and of the place where it is formed is facilitated by the fact that the cracks or fissures are of considerable size not only in length and width, but also in depth, as a result of which the rupture is not accidental in character and is represented not on a single section, but in the whole series of them.

Observing the successive changes in the structure of the root, we see that the size of the cracks increases with time. Thus, if in January they were poorly visible to the naked eye, then in February we could already easily see them on thin sections (see photos 2–5). For comparison of sections of frozen beet with sections of fresh beet, photographs 1–5 are presented. A longitudinal section of the root of frozen beet is shown in photograph 2. Here we clearly see ruptures, and they have a definite orientation corresponding to the arrangement of the vascular-fibrous bundles. Although in photograph 2 this is less noticeable than in the transverse sections of the root made in March, the purpose of this photograph No. 2 is above all to illustrate the very fact of rupture in the longitudinal direction of the root. Photographs 3, 4, 5 correspond to images of sections made across the root; the sections were made from roots of different diameter, at approximately the same place in the “root head.” As is evident in the photographs, the location of the ruptures in the fissures reflects the structure of the beet and indicates the circular arrangement of the vascular-fibrous bundles. Looking at these images, it is easy to notice that the ruptures are located between the vascular-fibrous bundles. Moreover, the greater the diameter, the more massive the root, the more ruptures there are. Thus, the photographs presented, of micro- and macroscopic specimens, definitively confirm the correctness of the proposition we have advanced.

But one cannot confine oneself merely to establishing the fact of tissue rupture; it is also necessary to consider the cause producing these ruptures. If the cause of rupture of the cell membranes were the expansion of the cell sap during freezing, then the ruptures would be disorderly in character. In that case, at best, the decisive factor would be the size of the cell and the degree of strength (elasticity) of its membrane. But if cell rupture depended only on the above-mentioned causes, then we would have had to observe a gradual formation of fissures, beginning with the rupture of only a few cells. At the be—

in those same preparations we could not observe this regularity. Examining carefully the membranes of their cells, cut in halves with a microtome knife, using the greatest magnification available to us (2250 times), we could not detect even signs of rupture either near the pores or away from them. The occasional injuries to the cell membrane that are observed we are inclined to explain rather by imperfections in the technique of preparing the slides than by the action of frost.

Thus, the more probable assumption is that the rupture in the form of a slit between the vascular-fibrous bundles arises not gradually, but rapidly and suddenly, and that the crack that has formed then gradually increases. What causes the appearance of the crack? To this question, it seems to us, the following answer is the more plausible: the cells distant from the vessels are the largest in size, and their walls are thinner. The intercellular spaces here are large and, being filled not only with liquid but also with gases, readily yield to compression. With expansion and change in the volume of the cells, owing to the unequal elasticity of the tissues composing the root, an uneven tension results, in consequence of which even displacements are possible. Whereas the vascular-fibrous bundles constitute, as it were, a “framework,” the interfascicular parenchyma is the place most favorable for tensions and ruptures.

In order to establish, at least approximately, at what temperatures and after what interval of time rupture occurs, beginning on 14 January and almost daily until 5 February batches of beet pieces measuring \(3 \times 1 \times 1\) cm were taken out and placed in an English-type meteorological box. Studying them, we came to the conclusion that rupture of the tissues, although in this case it also occurs, is considerably smaller in size and number than in whole roots. In general, the degree of injury from the action of cold is proportional to the mass of the root.

Studying the beet pieces taken outdoors on 14 January, we were able to observe the phenomenon of rupture only on 25 January, i.e., after 11 days. The period of time from 14 to 25 January 1929 is characterized by five-day periods as follows:

Fluctuation of minimum temperatures.

The ruptures in the above-mentioned slices reached still larger dimensions in the last days of February.

Passing to the results of the cytological study of the roots of frozen sugar beet, it is necessary to indicate that the greatest attention was devoted to the nucleus and nucleolus. The nucleus and nucleolus, despite their small size in comparison with the size of the cell, are the best reagents and indicators of the processes occurring under the influence of

cold on the physicochemical processes inside the cell. It must be thought that, as a result of a more thorough study of the nucleus, it would be possible to trace a gradual picture of the changes in this important organoid of the cell, and subsequently, on the basis of changes in the structure of the nucleus, to determine in what state the cell was at the moment of fixation.

The cytological investigation undertaken by us is in complete agreement with the data of Matruchot and Molliard, but certain details concerning the fate of the nucleolus, which escaped the observation of the above-mentioned investigators, were discovered by us by using the more complex and more advanced method of triple staining according to Blochmann.

Studying the cell of fresh beet, we observe the following picture: near the walls, stained blue, the nucleus is situated; its staining, like that of the protoplasm, is paler—bluish. The shape of the nucleus varies in the different tissues. In the interbundle parenchyma it is round, somewhat flattened at the sides; depending on its orientation, it appears round or somewhat elongated (see photograph 6). In the zone of dividing cells (cambium) and in the phloem, the relation between the shape of the cells and the shape of the nuclei is clearly observed. In elongated cells the nucleus is spindle-shaped (see photograph 6). In studying the preparations, we quite often encountered cells with two nuclei. Inside the nucleus there is a nucleolus; it is sharply separated on all sides from the mass of the nucleus by a light strip, a “courtyard.” This formation must be regarded as postmortem, arising during the treatment of the material. The nucleolus is always stained red.

In preparations made from beet subjected to the prolonged action of cold (from autumn until January 20), in addition to the aforementioned ruptures of tissues, we see that nuclei, as morphological formations, no longer exist. Instead of them, in various parts of the cell there occur shapeless pieces—flakes—of some substance staining bluish. We also do not find nucleoli, and the red staining is entirely absent.

If Matruchot and Molliard observed vacuoles in the nuclei of frozen and then thawed objects, then in our experiments, observing the same pictures about which the above-mentioned authors wrote, and studying roots subjected to more prolonged and strong cooling, we encountered the phenomenon of complete rupture of all nuclei (photographs 8, 9, fig. c, d, Table III). The nuclei of cells of smaller size with a large amount of protoplasm prove to be the best preserved. We can judge the influence of freezing on the nucleoli by the red staining characteristic of them. In our preparations we were able to observe (photographs 10 and fig. c, d, Table III) the disintegration of the nucleolus into finer parts. These particles had not yet lost the ability to stain red and stood out sharply against the blue, already shapeless background of the nucleus.

Our preliminary orienting investigation is far from complete: we have not yet precisely indicated the temperature limits at which rupture of the membranes of cells, nuclei, and nucleoli occurs; we also cannot yet depict the above-mentioned phenomena in the necessary sequence of changing pictures. The basic primary questions—the rupture of the membranes of cells, nuclei, and nucleoli—are for us an obvious fact. As has already been said above, in the cells

in fresh beet the nucleolus is stained red by safranin; in cells subjected to the action of frost, however, by Wasserblau dye it is stained a bluish color. This testifies to changes taking place within the nucleoli. The nucleolus becomes “acidophilic.” According to the most recent data, the change in the ability to be stained in other colors is explained (Romeis, Taschenbuch der mikroskopischen Technik, Allgemeiner Teil, 12th ed., 1928) not only by a chemical change, but also by a physical one—a change in density.

Studying sections of frozen, unfixed beet, we constantly observe air bubbles not only in the intercellular spaces, but also inside the cells. The fact established by us is in full agreement with Schaffnit’s data and indicates that ice forms, when roots freeze, both in the cells and in the intercellular spaces. Attempts, by means of microscopic investigation, to find a difference between the structure of the tissues of frozen beet subjected to the action of steam for 5–6 seconds and that simply thawed in a room at 15°C did not reveal any noticeable difference. A certain slight change in the tones of the staining can hardly be considered a sufficiently reliable sign of the changes that have occurred.

We must also dwell on the influence of methods of thawing. In the literature on this question there are the following indications. Buffon Du-Hamel (1737), Sachs (1870), and Preilleux (1872) considered that thawing is the decisive moment in the possibility of preserving life. This point of view is fully shared by practitioners as well. Maksimov, who strongly opposes the importance of various methods of thawing, nevertheless on pp. 291–292 and 299–300 of his work “On the Freezing and Cold Resistance of Plants” considers it possible to acknowledge that gradual thawing may prove salutary, since before the plant loses a considerable amount of moisture, the plasma will restore its impermeability, and the water from the intercellular spaces will again be absorbed by the cells. Senebier (1800), Göppert (1830, 1870–71), Künisch (1880), and others dispute the importance of thawing, believing that the rupture of tissues occurs during freezing. Muller-Thurgau, refraining from a definite judgment as to the moment of death—whether it occurs during freezing or at the moment of thawing—objects decisively to the entrenched opinion that a frozen plant can be saved by means of slow thawing. Besides these literary data, in the same work of N. A. Maksimov we find that the speed of thawing has a certain significance for apples (pp. 32–33). Comparing the facts cited above, we consider that thawing may have significance when an organism subjected to the action of cold undergoes certain changes, but these changes are a reversible process, i.e., with slow thawing the tissues and cells can assume their original normal structure. By thawing the same plants rapidly, we can cause new, deeper changes, and the organism ultimately dies. In saying this, we must of course take into account that with prolonged freezing such profound changes occur that it is impossible, by any kind of thawing, to restore the original structure and properties.

In our investigation we are dealing with beet roots that have remained for several months in the frost, at a temperature reaching as low as −40°C. There is no doubt that to “revive,”—to restore the ability to germinate and live—to these roots by any means whatsoever is ...

was a method—impossible. But this question does not fall within the program of our investigation. We are interested only in the question of how various methods of thawing affect frozen beet with respect to the preservation of its tissues and cells. For this purpose the material, in the form of pieces, was first subjected to freezing for from 2 to 14 days (in the period from January 14 to January 28) at temperatures from −12.0°C to −40.5°C, and was then thawed in air at \(t\) 2°, 10°, 15°; in water at a temperature of 2°, 12°; and in vapors—about 100°C; after the above-mentioned treatments the material was fixed. In addition, one batch of frozen beet pieces was immersed in a fixative, the temperature of which was 3°. The temperature of the fixative rose gradually, and the beet slices thus thawed slowly. Studying the preparations made from this material (using the methods indicated above), we found no noticeable differences and consider that the thawing methods we used had no effect on the preservation of the beet, which apparently remains the same as it had been before thawing. All the foregoing indicates that ruptures in sugar beet occur during freezing, and not during its thawing. It should be noted that the majority of biologists who have hitherto dealt with the question of the influence of cold on the plant were interested in the plant so long as it was alive, so long as it could be restored to life, and therefore in their experiments the organism was subjected to the action of cold for seconds, minutes, and hours; under the conditions of our experiment, however, the beet roots remained in frost, reaching −41°, for whole days, weeks, and months. We are interested in the structure not only of living but also of certainly dead tissue. Cells perish from cold even when their sheaths remain intact—this proposition is fully confirmed also by the data of our investigation, which showed, at the same time, that the further prolonged action of temperatures entails rupture of the tissues and a whole series of new changes in the structure of the cell that has already perished.

The fact established by us, that tissues rupture, does not refute the correctness of the most recent views. The object of study and the conditions of our experiment are entirely different, and the results of our investigation will be a supplement to what was done before us on this question.

CONCLUSIONS.

1. The onset of death of the cells of sugar-beet roots can be established by using the following signs:
   a) Disappearance of osmotic pressure, determined by means of macroplasmolysis;
   b) Darkening of the tissues of the beet root due to the activity of enzymes, whose action in dead cells is manifested quite clearly at zero degrees and at still lower temperatures.
   c) Structural changes in the nucleus and nucleolus.
2. Depending on the duration of the action of low temperatures, the tissues of the beet root rupture; in the nucleus and nucleolus initially [[unclear: text continues on next page]]

vacuoles, the number and size of which increase with time and, finally, the nucleus and nucleolus disintegrate into small parts; the substance of the nucleolus, which in living cells is stained red by safranin, upon their disintegration is stained blue.

1. Ruptures of the tissues, nucleus, and nucleolus occur when sugar-beet roots freeze, and not when they thaw.

2. The action of cold under the conditions of the experiments performed, at a temperature of about \(-9^\circ\mathrm{C}\), kills the plasma of the cells of the beet root already after 8 hours.

CONCLUSION.

The root of the sugar beet, when subjected under natural conditions of the Siberian winter to the prolonged action of steady cold without thaws, undergoes profound changes. In the root, ruptures appear in its parenchymatous tissue between the vascular-fibrous bundles. The size and number of these reservoirs increase with time. Masses of ice of pure water, initially appearing in the intercellular spaces, gradually increase with time, forming elongated accumulations that penetrate the body of the frozen beet root. Already by the month of January, under the action of the natural cold of the Siberian winter, the sugar beet is transformed into an entirely new material, unknown to European sugar production, sharply different in its biochemical, physical, and mechanical properties from ordinary sugar beet stored in an unfrozen state. The study of the phenomenon of plasmolysis, of the darkening of beet sections, and the study of structural changes in the nucleus and nucleolus of the cells of the parenchymatous tissue of the beet root make it possible to determine more precisely the dependence of the structure of sugar-beet roots stored in the open air on meteorological and climatic factors. This will make it possible finally to outline the best conditions for storing sugar-beet roots under the conditions of Siberian production. In addition, this method can shed light on the question of those limiting lowest temperatures at which sugar beet could be stored without freezing in regions with a milder climate, excluding the possibility of sugar beet freezing when stored in clamps.

Tomsk.

Explanation of the photographs and drawings

Photograph 1. — Transverse section of a fresh beet root.
Photograph 2 — Longitudinal section of a frozen beet.
Photographs 3, 4, 5. — Transverse section of frozen beet roots.

Photographs of Figs. 2–5. Made (0.8 natural size) from sections of beet roots previously thawed in air at a temperature of 15° C; they had been kept from autumn until February in the open air (in the city of Tomsk the air temperature during this period fell to −41.4° C).

The thickness of the sections, made by hand with a sharp razor, was approximately about 2 mm.

NOTE: The number and size of ruptures increase with the size of the roots.

Photographs 6–10 and drawings a, b, c, d, e — made from preparations prepared by the above-described method: fixative—Flemming’s strong concentration; staining according to Blochmann (thickness of sections from 8–16 microns).

The photographs were made with compensating eyepieces 10 and 20 and objectives 10 and 20 (Zeiss firm).

Photographs 6 and 7. — Fresh beet. There are no ruptures. Nuclei are visible in many cells; in some individual cells, 2 nuclei are present. The shape of the nuclei corresponds to the shape of the cells. The plasma, in an insignificant quantity, is located at the cell walls in the form of fine-grained, barely noticeable clots.

Photograph 6 — approx. 10 ocular; obj. 10, section thickness 8 microns.
Photograph 7 — approx. 15; obj. 10, section thickness 16 microns.

Photograph 8. Frozen beet (from autumn to 20/I). Each vessel separately and the vascular-fibrous bundles as a whole are preserved; considerable ruptures are noticeable in the zone of the interbundle parenchyma. Nuclei are absent (destroyed by cold).
Approx. 10, objective 10, section thickness 8 microns.

Photograph 9. Frozen beet (from autumn to 20/I). In the zone of the interbundle parenchyma a considerable rupture is noticeable. Nuclei are absent.
Approx. 15, objective 10, section thickness 16 microns.

Photograph 10. Frozen beet (from autumn to mid-December). In some cells, although the nuclei have already lost their shape, the chromatin has still been preserved in the form of shapeless clots, within which small red granules of the melted nucleolus are visible (in the photograph they appear as light points against the background of dark clumps—clots of the nucleus).
Approx. 15, objective 20; section thickness 8 microns.

Drawings a, b, c, d, e — made with the aid of an Abbé drawing apparatus. The granular substance of the nucleus is indicated by dots (on the preparations it stained blue); the nucleoli and their particles—clumps of varied form and size, produced as a result of the destruction of the nucleoli by the action of cold—are filled in with ink (on the preparations the substance of the nucleoli and their particles is stained red):

Fig. a, b, c — made from preparations photographed in photographs 6, 7.
Fig. c, d — made from the preparation photographed in photograph 10.
Fig. a, b — normal nuclei with 1–2 nucleoli from the root of an unfrozen sugar beet.
Fig. c, d — disintegration of the nucleus and nucleoli.
Fig. e — cell with a nucleus from unfrozen beet (relation between the size of the nucleus and the cell).

Fig. a, b, c, d — made with an immersion apochromat system 2 mm, Zeiss, and compensating eyepiece 20. Magnified 2250 times.
Fig. e — made with a Reichert 16 mm apochromat and compensating eyepiece 20. Magnified 375 times.

To the work of S. V. Lebedev and V. P. Chekhov.

a  b

c  d

e

[Illustration plate showing five specimens labeled 1–5.]

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10

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1. Blochmann’s mixture: water—100 cm³, Wasserblau—0.5 g, picric acid—0.25 g. ↩↩↩