Growth and Structure of Cotton Fiber 9 MATERIAL for the study of DONALD B. ANDERSON tlie litcrature. Obviously the results of investigators working with different cellulose deposition in cell walls, U. S. Department ofAgriculture and varieties grown under dissimilar condiNorth Carolina Experiment Station cotton fibers possess unique adtions will not be in agreement upon all v a n t a g e s o v e r other plant tissues. THOMAS KERR points. Cotton hairs are single-celledoutgrowths u. s. Department Of Agricultum* The study described in this paper is of e p i d e r m a l cells, and, since they Raleigh, N. C. based upon observations of the Mexican originate on, or very soon after, the Big Boll variety of American upland dav of flowerine. it is not difficult to cotton (Cossypium hirsulum) grown under field conditions trice their ent& growth history day by day. Wood and at Raleigh, N. C., during the summer of 1936. The same bast fibers, on the other hand, are embedded in a complex mass of tissue, the time of their origin is unknonn, and there general type of wall structure will be found in most other varieties, but the number of days during which fibers origiis no possible way of following their development at regular nate, the period of elongation, and the duration of secondary intervals. thickening of the fiber wall will not be comparable. The growth history of the cotton fiber falls naturally into two distinct phases: (a) a period of cell elongation and (b) TI32 most complete account of the origin and growth of a period of cell wall thickening. The cell wall formed during the period of elongation, the primary wall, is very differcnt cotton fibers has been given in the comprehensive studies of Balls (1-5) who worked upon Egyptian cotton (Gossypium in its clieiriicai composition and physical structure from the socondary wall which is laid down after elongation has ceased. barbadense). The early history of fiber development in the Mexican variety used in this study is in essential agreement The existence of decided contrasts between the primary and secondary walls in both chemical and physical properties, with the results reported by Balls. Each cotton fiber originates as an outgrowth from a single epidermal cellof theseed coupled with differenccs in the age of the fiber cell at the time of the formation of these walls, makes i t desirable to discuss coat. The first evidence of the formation of the cotton hair each phase of wall formation separately. is the appearance on the day of flowering of a slight swelling It is a common practice of investigators studying cotton of the outer wall of these epidermal cells (Figure 1A). The (Ievelopnients to tag the flowers and to collect their material swellings elongate rapidly and on the day after fiowering have at dated intervals thereafter. All cotton varieties do not already produced delicate tubular outgrowths, the young mature with equal rapidity, however, and decided differences fiber cells (Figure 1nj. The diameter of tlie mature cotton io behavior nray occur even when plants of the same variety hair is reached soon after it originates, but elongation of the are grown under different environmental conditions. Cotton cell continues for a pcriod of 15 or 20 days, when i t ceases fibers from bolls produced early in the summer will differ in abruptly (Figure 2). The period of elongation seems to be physical structure from fibers developed on the same plant determined by the time of year at which the flower opem and later in the season. These facts havc led to some differences possibly by environmental factors as well. Cotton fibers of of opinion among cotton workers nnd to some confusion in the Mexican variety attain a length of about 28 mm., Then
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period of elongation ends. A cotton hair is single cell, some 1200 times as long as i t is .. Se, that is attached to the seed only at its ;ie. Fiber origin is not limited to the day of flower:. Swelling and tubular enlargementscontinue be formed from the outer wall of epidermal $s for a period of about 10 days after flowerg (Figure 3). Those produced within 2 or 3 ,ys of the date of flowering give rise to the ,tion fibers of commerce, but those arising ter apparently produce the shorter hairs (fuzz tirs or linters) which form a dense tangled mat, kat adheres closely to the cottonseed. During the period of elongation the protoiasm of the cotton hair is enclosed only by the iin primary wall. This wall is commonly dcspated in mature fibers as the cuticle. Al~ thickness, the primary Tough only about 0 . 5 in iall contains at least two substances-pectic omuounds and ceuulose, and urobahlv waxes as ?ell: There is no evidence of the iresence of OF COTTON OVULEEPIDERMIS 4 D*us FIOURE3 (Left). CROSSSECTION AXTER OPENINO OF FLOWERS ( X 550) utin. AITO’KB indioate the yonos, fibers srising betwsen the bssee 05 the a i d s hairs. The presence of pectic substances in the prinary wall is indicated both by staining reactions (Right). WALLOF A &DAYFIBERA ~ E RTREATMENT FOR 24 FIGuRE H~~~~ IN BOILIKG md by solubility tests. The primary wall stains 0.5 PERCENTOXALIC ACID,FOLLOWED RY 24 HOURS IN ieeply with ruthenium red and is insoluble in BOILING 0.5 PERCENTAMMONIUM OXALATE ( X 366) xprammonia. When young fibers are treated The piotopissm hes been contraoted from the tip of the fiber. with hot 0.5 per cent oxalic acid followed by hot 3.5 per c e n t a m m o n i u m oxalate to remove sinm triiodide and 70 per cent sulfuric acid, the insolubility pectic compounds, the walls dissolve completely in cupramof the wall in fresh cuyrammonia, and the apparent isotropy rnonia. of the wall when observed between crossed Nicols have led It has been known for a long time that small amounts of several investigators to conclude that the primary wall of pectic substances are present in mature cotton fibers. Obyoung fibers is free from cellulose. Parr and Eckerson servations made liere indicate that these pectic compounds are (7) reported that cellulose first appeared in 5- or B-day-old restricted to the fibers. Hess, Tragus, and Wergin ( 2 1 ) found no ovidence of . primary wall. If cellulose until the fiber reached an ageof 36 days. Sakostsohimature cotton koff, Korsheniovsky, and Ilytikoly (16) found the first traces fibers are placed of cellulose in 13- to 15-day fibers and not generally present in ruthenium red, in the wall until 16 days after flowering. the primaq wall (cuticle) s t a i n s A NUMBER of investigators have snggested that t.he skeletal deeply while thc material of the primary wallis not actually cellulose but some secondary walls reclosely related substance. Balls and Hancock (6)referred to main c o l o r Ic s s. the primary wall as being composed of “pro-cellulose.” Although r a t h e Sakostschikoff and Xorshoniovsky (1.4) had a similar opinion. n i u m red is not Wergin (27) considercd that the primary wall of cotton fibers specific for pectic from the time of flowering up to 35 days contains no true cellu\ s u b s t a n c e s , no lose hut a new membrane material (Primilrsubsranr) which case is known in exhibits faint anisotropy. Sisson (16),on the other hand, was which n a t i v e able to obtain typical Debye-Scherer diagrams of cellulose in pectic compoundi: &day-old fibers hy removing the noneellulosic constituents have failed tostain of the %-all. with this dye. JI, These conflicting observations may be due to the fact that DAYS AFTER FLOWERING g e n e r a l , pectic the sinall amonnt of cellulose in the very young fibers is FIGCEE 2. RATEOF FIBER substances are effectively insulated by pectic substances. If the fibers are ELONOATION f o u n d i n large freed from pectic material by boil& with hot dilute (0.5 per amounts only i n cent) oxalic acid for 24 hours, followed by treatment with hot soft tissues-i. e., tissnes composed chiefly of primary walls. dilute (0.5 per cent) amnonium oxalate for a similar period, In hard tissues composed largely of secondary walls (wood, a coherent cellnlose skeleton remains’ (Figxire 4). This bast fibers, etc.) the pectic substances constitute a negligihle skeleton is doubly refractive iii Canada balsam as well as in fraction of the total wall material. It seems possible, therewater and alcohol. It is completely soluhle in fresh cupramfore, that pectic substances may be restricted t o primary monia, and it gives the characteristic cellulose reactions with walls in most if not all tissues. chlorozinc iodide and with potassium triiodide and 70 per cent The primary wall of the young fiber possesses a coherent sulfuric acid. It is still possible that the cellulose forming skeleton of cellulose from the first day of its appearance. It 6 It is ieeognirsd that this treatment for the removal of metic subatanaa does not respond clearly, however, to the usual microehemieal CBUBR~ eome degradation of the cellulose by forming hvdrooallulasa. test for cellulose in the early stages of its development. The Metura Gbem treatd zn thm way lose their teneile atrength and shoa absence of a blue color with chlorozinc iodide, or with potasnil”ei”“B slip plsnes. ?
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the skeletal framework of the primary wall is not identical with the cellulose of the secondary wall. It does give the characteristic microchemical reactions of cellulose, is anieotropic, and exhibits, according to Sisson (16),the typical x-ray patterns of cellulose. IDview of these facts, further evidence is desirable before a distinction is made between the cellulose present in the primary and secondary walls.
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of very delicate anastomosing threadlike strands of cellulose.
AU fibers do not show the presence of a transverse system of threads with equal clearness. Visual evidence of the existence of a transversely oriented system of strands is supported by the position of the addition and subtraction colo# produced when a gypsum plate Red I is inserted between crossed Sicols. It is also suggested by the paraUel extinction and
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COTTON H A I R STAINED C O N 0 0 RED AND PHOTOGHAPIXED BETWEEN CROSSED NICOI.5 A . Cellulase strande in wail of 15-dhy h i r . B. T I B ~ ~ Borientation T ~ B of oaliuiase threads in wsll 01 %day hair.
The cellulose present in the primary wall forms an open meshwork of fine threadlike strands that frequently anastomose. The absence of conspicuous double refraction in the young primary wall is probably due to the presence of relatively large amounts of isotropic pectic substances that infiltrate and surround the delicate meshwork of cellulose threads. The cellulose threads in the primary wall can be made visible by staining the wall with Congo red or chlorozinc iodide and examining between crossed Nicols (Figure 5A). These dyes apparently become directionally adsorbed upon the surfaces of the cellulose micelles (9),and the double refraction of the oriented dye molecules plus the intrinsic double refraction of the cellulose makes the cellulose strands appear prominently anisotropic. Balls (S) was tho 6rst to use this method upon young cotton fibers, and he discovered the prosence of two opposing systems of fine spirally wound threads of cellulose in t,he walls of 10-day-old fibers. The spiral threads were found to make an angle of approximately 70" with the long axis of the fiber. The writers were able to confirm this observation and extend it to very young fibers (2 days old). Two systems of spiral threadlike strands can be seen when untreated fibers are stained with Congo red or chlorozinc iodide and examined between crossed Nicols. The cylindrical form of the young fihers makes it possible to ascertain definitely that two systems of spiral threads exist in the same wall. Both right- and lefthand spirals may be seen when the microscope is focused upon the upper wall; they disappear in the upper wall as the focal plane is lowered and appear in the lower wall when the focus is established upon the lower wall of the fiber. There is evidence that the cellulose in the primary wall may have still a third orientation. When the stage of the polarizing microscope is rotated so that the long axis of thr fiber makes an angle of about 45" with the planes of tlie Nicols, a system of transverse strands of cellulose becomw visible in many fibers ( F i e r e 5B). As in tlie case of the spirals just described, this transverse system appears to be composed
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by the marked dichroism obtained when the long axis of the fiher cell is parallel with the plane of tlie polarizing Nicol. The three systems of h e threadlike strands that make up the cellulose framework of the primary wall seem to be uniform over the entire surface of the fiber. The striictural organization of the wall a t the tip of the fiber appears to he identical with that present near the base and 8t all intermediate points. No evidence has been found that the direction of the spirals changes so as to produce reversals such as are common in the secondary wall. There is nothing in the structure of the primary wall to indicate that i t controls or influences the Dattern of denosition of the later denosited secondary wall.' Although the structural uattern of the cellulose in the mimary waK persists throughbut the period of fiber elonpath, there is evidence that some cellulose is added to tho wall during this t i l e . The double refraction in stained walls of 15day-old fihers (Figure 5.4) is clearly more conspicuous than that of 2-day material (Figure 5B). The spiral strands in the 15-day fiber wall are broader and appear more as narrow hands in contrast to the fine threads present in the very young walls. Further, the walls of 154ay fibers give a definite blue color with strong chlorozinc iodide, which is extremely faint or entirely absent in the wall during the early stages of fiber elongation. It has been imnossible to determine whether the cellulose added to the pri&ary wall during the period of elongation has been deposited as a series of very fine layers that conform in their orientation to the pattern already present, or whether new cellulose micelles are added to the delicate threadlike st,rands already present in the very young wall. If definite layers are added to the wall during the period of elongation, they must he extremely thin, for tho entire wall of 13-day fibers bas a diameter of only 0 . 5 or ~ less.
THE first evidence of the secondary wall appears on about the sixteenth day after the opening of the flower. Previous to
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this time the wall of the fiber has shown no evidence of any structure, so far as can he determined by observation with the usual compound microscope (Figure 6A). The structure of the primary wall just described is visible only when the stained wall is examined between crossed Nicols. I n sharp contrast to the structureless appearance of the primary wall. the first secondary deposition stands out prominently when stained with chlorozinc iodide &s a layer of anastomosing strands that wind in a steep spiral around the inner surface of the primary wall (Figure 6B). This spiral makes an angle of about 2030' with the long axis of the fiber in contrast to the approximately 70" spirals of the primary membrane. The first secondary deposition appears suddenly and forms a layer that completely covers the inner surface of the primary wall. One of the most conspicuous features of the first layer of the secondary wall is the presence of reversals. Reversals are areas in the wall at which the cellulose threads change from a right-hand to a left-hand spiral or vice versa. The number varies within wide limits hut i t is common to find a t y or more on a single fiber. Two general types have been observed: (a) The commonest type of reversal is one in which the spiral strands simply change their direction by bending around in the form of an arc (Figure 6C); (b) the second type is one in which one set of spiral strands ends and a second system of spiral strands running in the opposite direction begins. The ends of the threadlike strands of the two systems overlap a t the place of the reversal (Figure 6 s ) . On the day following the initiation of secondary thickening, a second layer of spiral threadlike strands is laid down on the inner surface of tAe first layer. This second set of threads does not necessarily follow the pattern deposited on the first day. The points of reversals frequently occur a t different places, and therefore the direction of the spiral is often exactly thereverseof that in thefirstlayer of thesecondarywall Furthermore, the angle which this second layer of spirals makes with the long axis of the fiber is often different from that of the spirals in the first layer. In many areas of the wall, however, the patterns of the second layer do conform to those of the first layer. It is difficult to follow exactly tlie
pattern of subscquent deposition, but in view of the behavior of tbe fiber on drying and in response to swelling agents it is probable that a pattern is soon estahlished which is followed rather closely by the layers deposited later. It may be well to emphasize again the anastomosing character of this early deposition. The long fibrillar-like threads have a definite spiral path hut clearly branch and rebranch with one another. The pattern may be illustrated crudely by thinking of a fish net pulled strongly a t diagonally opposite corners. The threads which compose the spiral vary in length, however, and in diameter they grade down to the limit of microscopic visibility. After the initiation of secondary thickening, cellulose continues to he added to the inner surface of the fiber wall until a few days before the boll opens. I n cotton blooming early in the season, this is a period of a t least 25 days. In many bolls, however, deposition continues for a considerably longer period. When the boll opens and the fibers dry out, the wall becomes much twisted; Balls (8) showed that the direction of these twists (convolutions) conforms to that of the spirally wound threads that make up most of the cell wall. Where a reversal in the direction of the spirals occurs in the wall, a similar reversal in the direction of the convolutions is found. Undoubtedly, the convolutions present in the mature dry cotton fiber are determined by the organization of the cellulose micelles that compose the cell wall. The micellar pattern that controls the convolutions is not present in the primary wall and is finally established a few days after secondary thickening has begun. The Russian investigators Sakostschikoff and Korsheniovsky (24) recently gave considerable prominence to what they consider a structure of cellulose threads deposited upon the inner surface of the primary wall just previous to secondary wall formation. If cotton fibers 18 or 20 days old are stained with strong chlorozinc iodide, a coarse irregular network of blue lines often appears (Fignro 6D). As the reagents penetrate the wall of the fiber, the lines of the network widen until they merge together, leaving visible only the conspicuous spiral threads of the secondary wall. The behavior is exactly that which would occur if the reagent penetrated irregu-
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lar cracks in the primary wall and spread laterally in the secondary wall from these cracks. The primary wall does not stain deeply with cblorozino iodide, and the dark blue color arises only when the reagent penetrates into the secondary wall. It is probable, therefore, that the netlike system em-
Kerr (2s) recently confirmed the observations of Balls regarding the existence of a correlation between the number of lamellae in the wall and the number of days during which thickening takes place. According to Kerr, the wall of the fiber is a continuous matrix of cellulose and the laminated
phasized by Sakostschikoff and Korsheniovsky is not an actual cellulose deposit, as they claim, but merely a result of the irregular penetration of the chlorozinc iodide through cracks or thinner areas in the primary wall. Bolls developing from flowers that opened early in the summer of 1936 matured in North Carolina in about 45 days. The cotton fibers in these bolls grew in length for about 16 days and then thickened their cell walls for about 25 days. Bolls developing from flowers that opened late in the summer of 1936 matured in about 70 days. In t h e bolls the period of cell elongation waa only a few days longer than in the early bolls, usually 18 or 19 days. The period of secondary wall formation was enormously increased, however.
appearance in swollen sections is due to alternations of dense and less dense zones of cellulose. In contrast to Balls hypothesis of intermittent growth, Kerr suggests that cellulose deposition is a continuous process that varies in rate when temperature differences occur. When night temperatures arc low (below 20" C.), less deposition occurs and the dense zones of cellulose laid down during the day appear sharply separated in the swollen sections. When night. temperatures are high, deposition of cellulose occurs much as during the day and the dense zones of daytime deposition are not clearly separatrd. Under controlled conditions of temperature and light, the distribution and thickness of growth rings in the wall of the cotton hair can be varied a t will. When cotton plants are grown under constant illumination and at approximately constant temperatures (about 30' C . ) , no growth rings are formed in the fibers (Figure 7B). When the temperature is varied under conditions of continuous artificial light, growth rings are produced. Likewise when the temperature is maintained a t an approximately constant level, and tbe lights are turned off and on a t IZhour intervals, indistinct growth rings appear in the fiber wall. The relative importance of light and temperature RS factors in growth-ring formation iS under further investigation a t present. Although i t is possible to control the growth-ring pattern in the fiber wall, it has not been possible to influence the spiral threadlike structure of the wall or the presence of reversals. Summarizing these points briefly we have a picture of the structure of the cotton fiber as follows (Figure 8):
COTTOE fibers produced early in the season can be distinguished from those that arise late in the summer by swelling the cross sections in suitable reagents. When swollen, the cross sections reveal a striking lamellation (Figure 7 A ) . This has been known for many years, but Balls (2) was the first to correlate the presence of these lamellae in the wall of the fiber with the number of days during which the wall of the fiber increased in thickness. Noticing that the cotton plants ceased growing during the hot afternoon periods, Balls assumed that the fibers likewise stopped the process of wall thickening a t this time and resumed growth during the night and morning. The lamellae were therefore considered t o be evidence of periodic, discontinuous growth. In early-season fibers the growth rings are relatively broad, and a t times each ring may reach a diameter of approximately 0 . 3 0 ~ . The rings formed in fibers that develop late in tbe season are thinner and rarely exceed 0 . 1 4 in ~ thickness. The width of the growth ring is exceedingly variable even in fibers from a single boll, but in general, late-season fibers have B large number of narrow growth rings whereas early season fibers have fewer and wider r i n e .
tions: (3a Eat right-hand spiral, (b) a h t Iifbhand spiral, and probably also (c) B transverse position. All three systems aeem uniform over the entire surface of the fiber cell.
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INDUSTKIAI, AND ENGINEERING C11GMISTRY
2. A secondary wall composed of many lamellae of cellulose. The lamellae are not scparated from one another by noncellulosic substances but represent. dense and less dense areas of cellulose. The layers are formed of systems of s irally wound branching threads, and the direction of the spirafis reversed at frequent intervals. 3. FrequentlS- the pattern of spirals first appearing in the seconds wall is not similar to that in subsequent layers of the wall. d 8 t of the layers of the wall, however, follow a pattorn that is estsblished soon aftcr secondary thickening has begun.
THAT the valuable properties of cellulose are more closely related to the state of its molecular aggregation than to its molecular size is becoming increasingly apparent. The importance of this subject warrants some disoussion of the minute physical organization of the wall of the cotton fiber. Two contrasting hypotheses have assumed some prominence in this country in recent years. One of these, the so-called micellar hypothesis, states that the long cellulose chains are aggregat.ed roughly parallel to one another into indefinite bundles or micelles. These micelles are bound together by overlapping cellulose chains that extend from one micelle to another. The micelles are assumed to have a definite orientation in the cell wall and because of this orientation to be responsible for the optical and physical properties of the wall. The second hypothesis which we shall term the “ellipsoidal particle” hypothesis, assumes that units of cellulose of visible size originate in the protoplasm of the cell. The hypothesis assumes that each particle of cellulose is coated with s m e cementing substance, presumably of pectic nature, and t,hat while in the protoplasm, linear rows of cellulose particles become cemented toget.her to form clisins of variable length. These chains of cellulose particles are assumed to migrate through the selectively pemcable membrane that limits the protoplasm, to the cell mall where they beoome ceniented by pectic or other cenienting substances to the existing wall of the cell. The “ellipsoidal partiele” hypothesis vas first foriniilated as a result of a study of the growth of the
