Polarizing Microscopic Study of the Origin and Growth ofe tinction Bands in Native Cotton Fibres

Transcription

1 Indian Journal 'OfTextile Research V'Ol.II, March 1986, Pp.I-6 / Polarizing Microscopic Study of the Origin and Growth ofe tinction Bands in Native Cotton Fibres A V MOHARIR Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi II 012, India B C PANDA Division 'OfAgricultural Physics, Indian Agricultural Department and V B GUPTA Research Institute, New Delhi , India 'OfTextile Technology, Indian Institute 'OfTechnology, New Delhi , India Received 7 October 1985; accepted 22 October 1985 Native cotton fibres in never-dried and dehydrated states show under crossed polarized light characteristic extinction bands along their lengths. The cause 'Ofthe 'Originand growth 'Ofthese bands is still unresolved. This p per reviews critically the published literature in the light 'Ofthe present author's experimental observations 'Onthe 'Originand g owth 'Ofextinction bands Some pl-ausible mechanisms for the 'Origin'Ofthese bands from 'Ourobservations with a polarizing icroscope 'Ondeveloping cotton fibres 'Ofknown ages are discussed in detail. The classical hypothesis 'Ofreversing spirals 'Ofcellulose fibrils in the secondary layers is refuted to explain the 'Origin'Ofthe extinction bands.. Keywords: Cellulose fibrils, Cotton fibre, Extinction bands 1 Introduction Cotton fibre has a fibrillar structure and the bulk of its cellulose is laid in the secondary diurnal growth layers l - 9. These growth layers have been shown to arise as a result of diurnal temperature cycles during the growth period rather than due to changes in illumination cycles as shown earlier 9-2'O. Even cotton fibres grown under constant illumination and temperature, though without discernible growth layers, show extinction bands and their cellulose also has been shown to be fibrillar+". Cellulose fibrils in the individual secondary layers form a spiral about the fibre axis 1,2,4, Some workers believe that the orientation {)f these fibrils in the.successive secondary diurnal growth layers to be similar, whereas others believe that the individual secondary growth layers can both be lefthanded and right-handed and also could differ in spiral structure, cellulose chain length and crystallite sizes 1,4,2' , Some workers 1,2,9,25,26,35,4'0-44 have argued that in each layer the sense of the helix reverses several times along the length of the fibre: These so called reversal points are identified as extinction bands in a polarizing microscope under crossed field. Several electron micrographs of fragments of secondary layers of cotton fibres by negative staining, shadow casting techniques and by surface replicas. have however not shown any clear evidence of the existence of an abrupt change in orientation in secondary la ers 13,35, Parallelylaid cellulose fibrils only re however seen. Some evidence of cross-orientatio s in successive secondary layers deposited in definite w ve patterns in developing cotton fibres have been r ently shown by freezefracture replica technique i a transmission electron microscope+", Extinction bands have een mapped by several investigators 1,16,29,33,38,49,53-55, and their frequency is reported to be influenced y genetic 32,53,56-58 and environmentai 1l - 13,5'O,53,58 59 factors and by irrigation+". The number of e tinction bands has been shown to be more in Gossy ium barbadense varieties than in the Gossypium hirsu um, Gossypium arboreum, and Gossypium herb ace m species of cotton fibres 22,28,55,6'0-62. Positio s of extinction bands are again shown to be as weak points by some workers 63-68, but as s rong points by some others 65, If the revers I points are weak points, their increased number in ossypium barbadensee+"? varieties cannot be easily re onciled with higher fibre strengths of the fibres of this species 55,61,7'O. The negative correlation betwee the number of extinction bands and the strength of otton fibres observed by some workers'p may be misleading because the.strength data on these fibr varieties were taken by them from a general bookie 63 of fibre characteristics, rather than from the actual easurements on the same fibres used for counting ext nction bands.

2 INDIAN 1. "FEXT. RES., VOL. II, MARCH 1986 Reversals of cellulose fbrils in individual secondary growth layers are believ d by some 32,7i to coincide exactly or nearly so. If h wever true, the intensity of blackening of extinction bands should progressively increase with the depositi. n of each secondary diurnal growth layer. No such re ort exists, nor have our own observations reported b low shown any such effect. Superposition of revers Is in individual secondary growth layers therefore a pears to be only speculative. In a system like the c tton fibre, where internal dimensions are changin with each deposition of diurnal-secondary growt layer 39,73, such a possibility seems doubtful. Reversal in secondary growth layers are again different from the occasional reversals in external convolution twi ts on cotton fibres, and yet coincidence in tensile ractures with reversals in morphological studies with a scanning electron microscope and a olarized light microscope= have been shown. Ho ever it has been clearly demonstrated 50 that sue acoincidenc~ 'i's limited to only the plane of obse vat ion and should not be mistaken as a morpho log calor structural coincidence. The evidence in resp ct of the hypothesis of reversibility of cellulose fibrils in secondary growth layers, therefore, contin es to be derived from the polarizing microscope. although.riot without doubtsii,7s X-ray diffraction patt rns taken by Wakeham et al. 70 of single fibres at the reversal position ha ve shown a high degree of orie tat ion and crystallinity. A comprehensive review of tructural reversals and their relation with mechan cal properties has been published": Several attempts have e~n made over the years to explain the origin and gr wth of extinction bands, and yet the exact cause andinanner are not definitively known. In the earlier 'p blications, extinctions have been attributed to the resence of pits in the cellwallis. Kuhn, as quoted by Balls 2, ascribed them to the mystic gyration of the protoplasm in the drying cell, and Balls? explained it a due to the movement of the growth centre within t e fibre, during secondary cellulose deposition. Iyen ar 78, observing moist fibres from ripe cotton bolls, C und the reversals to appear midway between the adj cent bends of the fibre. Each flattened portion develo s into a structural reversal; more often than not t e bend is also a reversal. However, none of these xplanations is convincing in the light of experimental observations. Whatever may be the eal cause of the origin of the extinction bands in cottor fibre, it is clear that these are formed during the secon ary thickening-period. It is also now definite that. t e cellulose fibrils, find their orientation in individual econdary growthlayers or in association with severa underlying layers' have a bearing on the origin of extinction bands. How and under what conditions the extinction bands should appear, need explanation. In this study, we have made attempts to explain the origin of the extinction bands in the light of some of our observations on developing cotton fibres of known ages from the dates of flowering, with the help of a polarizing microscope. 2 Experimental Procedure A large number of flowers in a Gossypium hirsutum variety 'Bikaneri Narma' were tagged for a week. Green cotton bolls of known ages were collected in double-distill-ed water in a stoppered bottle. A few drops of chloroform were added to prevent bacterial growth. Fibres were carefully unlocked under water by cutting open the green bolls, and were examined in wet condition on a Carl-Zeiss polarizing. microscope under crossed polarizer and analyzer condition. 3 Results and Discussion Fibres from 6,7-day-old bolls showed complete extinction from the tip to the base under all orientations in the crossed field, which is a characteristic of an isotropic fibre. Photographing these fibres was rather difficult for lack of sufficient transmission of light through fibres. The first result presented in Fig. 1 is a photograph of fibres collected from 14-day-old boll after flowering, as seen under a crossed-polarized light. The body of these fibres is dark and remains so even if the specimen stage is rotated, indicating that the fibres are essentially isotropic. Only the wall edges are seen illuminated and this is apparently an edge effect. The brightness in the body of the fibres generally increases with age and the clear distinction between the edges is lost after about the 20th day from flowering as seen from Figs 2 and 3. The first dark extinction band was seen in a fibre from a 21-day-old boll. Fibres Fig ~day-old fibres of Bikaneri Narma cotton varietyunder crossed-polarized field 2

3 MOHARIR et af.: ORIGIN AND GROWTH OF EXTINCTION BANDS IN NATIVE C TTON FIBRES collected from bolls on later days showed the appearance of more bands and the interband spacings became proportionately smaller for fibres from bolls collected still later. The appearance of new bands between the existing ones would be expected to result in this decrease of band spacing. Similar observations were made by Betrabet et al. 30,54,58,60. The spacings were generally not periodic, although in some fibres statistically regular spacings were observed. Randomness in extinction band spacings has also been shown by several workers 1,9,33,54,79 and-also a fall in the number of extinction bands per milimetre in the second picking of fibres, which Iyengar." attributes to the rise in the atmospheric temperature with the advance ofthe season. This decrease in the number of extinction bands in fibres of second picking may be due to the shorter boll maturation period (secondary thickening period) offered by the place of growth to the bolls from late flowers, owing to the advancing season. Raes et al. 66, however, observed a statistically significant periodicity in extinction band distribution and they presumed this to be inherent in the generation of extinction bands. Figs 4 and 5.show some illustrations of the extinction band spac ngs. It is therefore clear from the above observations that the formation of the extinction bands takes place uring the secondary wall thickening period. A definite correspondence between the number of days of second ry thickening period and the number of growth layers as been seen 5,9,2Q,53.It is also indicated that the perio of secondary thickening continues for well over 30 ays depending upon the environment. A prolonge range of secondary thickening periods (boll aturation periods) for Gossypium barbadense cotto from 51 to 74 days has been observed for locations n Egypt and Sudan, and this' has been ascribed t the differences in air temperatures compared t the other economic zones 81,82. Schubert et al. 3 and ltoh 84, however, indicate an overlapping f secondary thickening period to some extent over t e elongation phase, from their studies with an e ectron microscope on developing cotton fibres. Sin e in general the length of the fibres is determined before the secondary thickening begins, an in teres ing close correspondence between the mean fibre len ths per unit temperature cycles for the secondary t ickening period and the inter-reversal distance for the fibre of Gossypium. hirsutum species reported in the published litera-. Fig day-old fibres of Bikaneri Narma cotton variety under crossed-polarized field Fig day-old fibres of Bikaneri Narma cotton variety under crossed-polarized field Figs 4 & 5-Some examples of t e distribution of the extinction bands in co on fibres 3

4 INDIAN J. TEXT. RES., VOL. II, MARCH 1986 ture60,85, has been seen y Moharir et al. 50,59. These observations implied t at the diurnal secondary thickening periods ma be responsible for the formation of extinction ands. Whereas the existenc of cross-oriented cellulose fibrils in the primary w lls of cotton fibre has been generally accepted and s en", the evidence for abrupt change in-the direction 0 the fibrils laid in the walls of secondary layers is not y t established. On this subject there are various school of thought. Some workers believe that the fibrils a laid parallel to each other without any change in di ection. Others consider that the fibrils are deposited i Sand Z spirals in successive secondary layers. Yet oth rs believe that the spirals are deposited as the S spiral 0 ly. The spiral angle in all the successive secondary gro th layers, irrespective of the species of cotton, is bel eved by some 26,27,31 to be constant at 22. Others elieve that the spiral angle progressively decreases towards the core of the fibre21,32,37, Inc ease in refractive index for n II with respect to n1.in a fibre refractometer has been attributed to decreasing piral angle and to variations in spiral arrangement among different fibres or segments of the same fi reo Duckett and Ramey?", however, believe that the e is a rapid decrease in spiral angle in the outer di rnal layers, after which a constancy in spiral angle is approached. In the context of the discussion above, the most important question that eeds to be answered is : in the absence of abrupt spiral r versals in direction, how can the extinction bands appe r in a crossed polarised light? To answer this question the following mechanisms are considered and discu sed. 3.1 Mechanism of Formation f Extinction Bands The polarizing microsc pic studies have shown that the transverse extinction ands statistically increase in number with each diurn I layer deposited during the thickening of the cell wal, and that the conditions for origin of extinction band are actually formed during this period of a develo ing cotton fibre. Although many workers believed" that there are differences in the cross-sectional dimen ions of cellulose microfibrils from primary and second ry walls of cotton, Willison and Brown+' found no di ferences in the dimensions of fibrils from the freeze-fra tured replicas of these walls in developing cotton f bres using a transmission electron microscope. hey however found the individual microfibrils a d bundles of microfibrils in secondary layers deposit d in definite wave patterns with definite amplitude, avelength and relative phase differences. With this knowledge, here are several possibilities in which the mechanis of the origin of extinction bands may be worked ou. These are discussed below The spirally deposited cellulose fibrils in each individual secondary layer may be oriented parallel to the fibre axis at extinction points. In considering this possibility, we are required to revert to the classical hypothesis of the cellulose fibrils abruptly changing their sense of orientation from left to right and while doing so, they align themselves in an almost parallel position to the fibre axis and produce an extinction band. It is however very difficult to imagine a whole cylindrical di~rnal layer changing abruptly in orientation during its deposition. Also, it is difficult to conceive of such patterns in cellulose deposition being copied in toto in the same location in successive secondary layers deposited, particularly in a dynamic system such as cotton whose internal dimensions are changing with each secondary layer deposited. Here therefore two conditions arise. If the reversals in successive secondary layers deposited were to coincide with the reversals of the first ever secondary layer, then the number of the extinction bands seen along the entire length of the fibre must be determined in full number in the very first secondary layer deposited, and the successive layers merely copy them by exact superposition. Experimental evidence and our own observations reported in the early part of this paper however have shown that the number of extinction bands increases after the 21st day of flowering. Secondly, if we assume that the reversals in every individual secondary layer deposited do not coincide with each other but are established separately, or marginally ahead of the ones in previous layers, the whole fibre should appear dark under crossed polarized light or at least the extinction bands should diffuse into very wide zones. None of these two possibilities has been experimentally observed and therefore this postulate in explaining the origin of the extinction bands also seems implausible Formation of isotropic zones in the secondary layers along the length of the fibre. Assuming that the spiralling cellulose fibrils in increasing orientation from the outermost to the innermost sedondary layers create totally isotropic conditions at periodic intervals along the fibre through small cross-sectional matrix of the fibre, one can see extinction bands at such isotropic locations under crossed polarized light. Such bands arising out of the isotropic disposition of cellulose fibrils between superposing secondary layers must necessarily stay in their positions without change in intensity under all orientations of fibre in crossed polarized field. This is however not experimentally true since the extinction bands alternate from dark to bright on rotating the stage of the microscope. The conditions of isotropy 4

5 MOHARIR et al.: ORIGIN AND GROWTH OF EXTINCTION BANDS IN NATIVE TTON FIBRES between layers does not therefore seem to be the cause for the origin of the extinction bands in cotton fibres Formation of conditions for complete extinction may be a localized phenomenon between two successive or intermediate secondary layers in the upper half or the lower half of the cylindrical fibres, when observed vertically in a two-dimensional plane in a crossed polarized field. If this be true, and in view of the fact that the secondary layers are hot uniform in thickness along the cross-section of the fibres 5,6.48,87, it implies that cotton fibres at extinction points in one plane of observation should produce no extinction when observed orthogonally. Our observations of extinction bands in several cotton fibres in such mutually orthogonal views, on a specially designed axially rotating goniometer, show that the extinction bands stay in their positions along the entire crosssection ofthe fibre on rotation about the fibre axis. The formation of the extinction bands is therefore not a localised phenomenon between a few intermediate superposing secondary layers but is disposed through the entire circular cross-section of thel fibres. The above three arguments leave only one possibility on the origin of the extinction bands as discussed below Freeze-fractured replica of secondary layers of developing cotton fibres'" have shown that the individual cellulose microfibrils and bundles of microfibrils are deposited in definite wave patterns with a definite amplitude, wavelength, and phase differences between them. These wave patterns made by the bundles of microfibrils contribute to the variable orientation of the anisotropy revealed by polarized light studies. These bundles of wavy fibrils within an individual secondary layer or in co-operation with a number of underlying or overlaid layers form a narrow region of crystallized matrix in which the orienting bundles of cellulose fibrils are held parallel or nearly parallel to the fibre axis, thereby producing extinction bands. If however these bundles had been laid exactly parallel, the extinction bands would have been expected to change from dark to bright at each 90 rotation in a crossed polarized field. Close observation of extinction bands shows that they alternate from dark to bright at each rotation of the stage of the microscope. This means that there is a cross-oriented crystallized matrix of cellulose fibrils at the extinction bands. Such orientations may therefore be possible between the successive secondary layers. The little periodicity observed in the distribution of extinction bands must therefore be associated with the periodic wave patterns in which the cellulose fibrils are deposited within the secondary layers and their periodic in-phase superpos tion. Assuming that the wave patterns of the deposit d cellulose fibrils in every secondary layer remains the arne, the wave patterns in the freshly laid layers woul be laid slightly ahead of the waves in the preceding I yers owing to the reduced diameter of the fibre. As a c nsequence, the number of extinction bands would be xpected to be more in the upper half of the developi g cotton fibre along its length than in the 10 er half. Experimental observations of increased frequency of extinction bands in the upper hal ves of otton fibres 5 support our hypothesis on the origin of cotton fibres. xtinction bands in native 4 Conclusion We refute the classical h pothesis that the reversing cellulose spirals in seconda y layers of cotton are the cause of the origin of extin tion bands. The origin of these bands is therefore a phenomenon between secondary layers deposite in a developing cotton fibre. Since the duration of the deposition of the secondary layers depe ds greatly on several environmental and growt conditions, besides the genetic factors, varietal c aracterization of cotton fibres based on the freque cy of extinction bands is almost impossible. Prolo ed secondary deposition periods for the varieties of the long-staple Gossypium barbadense species 82,83 als explain why the fibres of this species have an incre sed number of extinction bands seen on them. Also, since the extinction bands arise as a result of the orde ed regions in the matrix of the cotton fibres, their ncreased number should correspond to higher stren ths of the fibres as has been observed by some worker 4,22,28,55,60. 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