GIFT OF

«•«*?

ABSCISSION OF FLOWERS AND FRUITS IN

THE SOLANACEAE, WITH SPECIAL

REFERENCE TO NICOTIAN A

A THESIS SUBMITTED IN PAETIAL SATISFACTION OF THE EEQUIEEMENTS FOE THE DEGEEE OF

DOCTOE OF PHILOSOPHY AT THE UNIVEESITY OF CALIFOENIA

BY

JOHN NORMAN KENDALL

MAY, 1917

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Vol. .5, No. 12, pp. 347-428, 10 text figs., plates 49-53 March 6, 1918

ABSCISSION OF FLOWERS AND FRUITS IN

THE SOLANACEAE, WITH SPECIAL

REFERENCE TO NICOTIANA

BY

JOHN N. KENDALL

UNIVERSITY OF CALIFORNIA PRESS BERKELEY

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Vol. 5, No. 12, pp. 347-428, 10 text figs., plates 49-53 March 6, 1918

ABSCISSION OP FLOWERS AND FRUITS IN

THE SOLANACEAE, WITH SPECIAL

REFERENCE TO NICOTIAN A

BY

JOHN N. KENDALL

CONTENTS

PAGE

I. Introduction 348

II. Summary of the literature 350

III. Technique 361

IV. Histology and cytology of the pedicel 363

1. Histological and cytological condition of the mature pedicel 363

2. Development of the separation zone in Nicotiana and Lycoper- sicum 367

3. Increase in size and development of mechanical tissue in the pedicel of Nicotiana and Lycopersicum 369

V. The process of abscission 371

1. General description of the process in several genera 371

2. Method of cell separation 376

VI. Abscission of the style and corolla 383

VII. Time of abscission 385

1. Keaction time 385

2. Abscission time 396

VIII. Experimental induction of abscission 397

1. Induction by illuminating gas 397

2. Action of acids on the separation layer of Nicotiana 404

3. Induction by mechanical injury 406

4. The ability of certain species to throw off pedicels from which

all the floral organs have been removed, as related to the induc- tion of abscission by mechanical injury 410

IX. Summary 411

X. Conclusion 415

XI. Literature cited 418

XII. Plates .. 420

374794

-

? *•; • : %«• * / . • •»•«•

348 University of California Publications in Botany [VOL. 5

INTRODUCTION

Although it is a matter of common observation that many plants are capable of detaching portions of the body, the underlying cause and the actual mechanism which bring about such separation are only slightly understood. The process has often been described as one of self-pruning by which the plant rids itself of useless portions of its body. Since abscission is sometimes confused with exfoliation, it seems desirable here to distinguish definitely between these two phenomena. It can be said that, in general, exfoliation is preceded by drying and death of the part to be cast off and that actual separa- tion of the organ is accomplished by a mechanical break through dry, dead tissues. Abscission, on the other hand, is usually not preceded by drying and death of the organ concerned and its detachment is accomplished by a separation along the plane of the middle lamellae of active living cells.

Abscission may be either axial or lateral. Axial abscission includes the abscission of portions of stems, shoots, entire flowers or fruits. Lateral abscission includes the abscission of leaves, petioles, sepals, petals or styles. Considerable attention has been given by investi- gators to the abscission of flowers because of the theoretical detriment to crops caused by the fall of the flower before the fruit is formed.

The cause of leaf-fall in deciduous species is connected with peri- odic changes in the physiological condition brought about by changes in the environment. In the case of some herbaceous plants and occa- sionally in trees, sudden changes in environmental conditions result- ing in a loss of physiological equilibrium often cause the throwing off of leaves, flowers or even small shoots. In certain species, any- thing which tends to loss or completion of function within or peculiar to an organ causes the organ to be thrown off. Thus, staminate flow- ers are commonly thrown off soon after anthesis and pistilate flowers generally fall when fertilization is prevented. Similarly, certain species — e.g., Impatiens Sultani and Mirabilis Jalapa — throw off por- tions of their stems which have been rendered useless as a part of the conducting system because of injury or removal of distal buds or leaves.

The following definitions of terms, which will be used throughout this paper, are made necessary because of a notable lack of uniformity in their usage by various investigators who have dealt with abscission.

1918] Kendall: Abscissi-on of Flowers and Fruits in Solanaceae 349

1. Abscission is the detaching of an organ by the separation of actively living cells at or near its base.

2. The separation layer (Mohl's Trennungschichte) is the layer of cells the components of which will separate from one another at abscission.

3. The separation cells or absciss cells are the cells that make up the separation layer.

4. The separation zone is the general region through which abscis- sion takes place and usually is largely proximal to the separation layer.

A preliminary account of abscission in Fx species hybrids of Nico- tiana has already appeared (Goodspeed and Kendall, 1916). The present study represents an amplification of this investigation and its extension to other species of the Solanaceae. It is particularly con- cerned with the following: (1) the position of the separation layer; (2) the origin of the separation layer; (3) the cytology of the separa- tion layer; (4) the process of abscission, including (a) a description of the appearance of the separation layer in consecutive stages of the process and (b) the method of cell separation; (5) the time occupied by abscission, including (a) the time between the application of the stimulus and fall (reaction period) and (b) the time involved in the actual process of cell separation (abscission period) ; (6) experimental induction of abscission.

Although the investigation reported here is largely a morpholog ical one, the results of the experiments on the method of cell separa- tion, the time of abscission and the induction of abscission seem to have a distinct physiological significance as well.

350 University of California Publications in Botany [VOL. 5

SUMMARY OF THE LITERATURE

Since the literature on abscission is rather voluminous, it seems best to present the following discussion under several different head- ings corresponding, to a certain extent, with the six main topics of interest mentioned in the introduction. The summary below is largely confined to the literature on axial abscission, although that on lateral abscission is considered in so far as it has a direct bearing on the most important aspects of the abscission problem.

1. HISTOLOGY OF THE PEDICEL

a. POSITION OF THE SEPAEATION LAYER

Hoehnel (3880), discussing the fall of catkins in Populus and Salix, locates the separation layer at the base of the catkin. The gen- eral region at the base of the catkin, in the distal part of which the separation layer is located, he calls the "separation zone." In Salix, actual separation occurs in the separation layer, but in Populus it occurs in the parenchyma entirely outside the separation layer. According to Balls (1911), the separation layer in the cotton flower is located at the base of the pedicel. The layer is located by Hannig (1913) at the base of the pedicel in Nicotiana Tabacum, N. rustica, N. accuminata, N. sylvestris, Datura, and Atropa, and at the tip of the pedicel in Nicotiana Langsdorffii, Salvia Aloe, Cuphea, and Gasteria. He finds it occurring at the middle of the pedicel in Impatiens Sultani, Solanum tuberosum, Lycopersicum, Asparagus, and Begonia. Gort- ner and Harris (1914) and Lloyd (1914&), working on the abscission of internodes as the result of injury in Impatiens Sultani, locate the separation layer at the first node below the injury and just above the axillary bud. Occasionally, according to the latter investigators, ab- scission may occur at the second or third node below the injury and in these cases the buds at the first or second nodes seem to be abortive.

The separation layer, according to Hannig (1913), may occur at the base of the complete inflorescence in Impatiens and Oxybaphus. According to Lloyd (1914a), the separation layer occurs at the base of the pedicel in cotton and at the base of the ripened ovary in grape "shelling." In the abscission of internodes and tendrils in Vitis and Ampelopsis, Lloyd (19140) locates the layer near but not exactly at the base of the internode. A peculiar case illustrating the result of displacement of the stem on the location of the separation layer is

1918] Kendall: Abscission of Flowers and Fruits in Solanaveae 351

discussed by Lloyd (1914a) for Ampelopsis and Gossypium. In the latter, abscission, in the abnormal case, occurred down the internode at the base of the pedicel. This is explained as the result of a dis- placement during growth by which part of the pedicel becomes united to the stem.

Occasionally, grooves or swellings are noticed at the base of the organ being abscissed where they correspond more or less" exactly to the general position of the separation layer. Examples are given by Hannig (1913) for Lycopersicum and Solanum tuberosum and by Balls (1911) for Gossypium. Abscission may occasionally occur, according to Lloyd (1914a), above a small bract. According to these latter investigators, there is more often no external indication of the layer. Frequently, grooves bear no relation to the layer because in many cases of this kind (Hannig, 1913, for Brunfelsia) separation occurs a short distance distal to the groove.

From the above brief summary it is evident that in the case of axial abscission the separation layer is located at or near the base of an internode. Apparent exceptions are reported by Hannig (1913) in which it is seemingly located at the middle of an internode. It seems probable that a more critical re-examination might reveal the fact that even these exceptions accord with the general rule. In these cases, for example, the pedicel of the flowers in question might be composed of two internodes.

6. ORIGIN OF THE SEPAEATION LAYEE

Kubart (1906) states that the occurrence of the separation layer in all tyes of abscission may be explained in one of the three following ways: (a) the separation layer is preformed and represents simply a portion of the primary meristem which has remained in its original active state; (6) it represents a secondary meristem; (c) the primary meristem may function directly as a separation layer. The differ- ence between a and c is only a difference in time, c being added to explain the origin of the separation layer in abscission of very young, embryonic tissues. In a, the separation layer is present at the base of the organ from the start of its development, but in & this layer has to be formed by a secondary meristem before abscission can occur. In a, cell divisions are not normally found preceding abscission, but in & and c they are. Mohl (1860), working on the fall of the flower in Aesculus, Pavia, Lagenaria, Cucumis, and Ricinus, states that the separation layer in these forms is of type 6. Throughout his entire

352 University of California Publications in Botany [V°L. 5

work Mohl gives the general impression that it is necessary for a sep- aration layer to be formed from a secondary meristem before abscis- sion can occur. Wiesner (1871), working on leaf -fall in general, observes that the separation layer is not generally of type b, as Mohl believes, but more often of type a. According to Becquerel (1907), the separation layer is formed in the pedicel of Nicotiana from a sec- ondary meristem (type b). In the cotton flower Balls (1911) finds that the separation layer is of type Z>, but according to Lloyd (1914a and 1916&) there is doubt as to this conclusion, since in the case of very young cotton flowers in which abscission occurs very suddenly, he finds only rarely that cell divisions do not precede abscission. Hannig (1913), for flower-vfall in general, states that a separation layer of type a is always present but in certain species a secondary layer of type b may also be formed, through which separation may or may not occur. Hannig, differing from Becquerel ( 1907 ) , points out that the separation layer in Nicotiana is of type a. Lloyd (1914a) and Loewi (1907) indicate that in general a layer of cells through which abscission is possible is more often of type a than of type b. They believe, however, that the separation layer is not a definite morphological structure but represents merely a physiological con- dition.

c. CYTOLOGY OF THE SEPAKATION LAYEE

Mohl (1860) describes the separation cells in the flower stalk as young, active, small cells which generally contain no starch. He also states that in most cases cell divisions are characteristic of the sep- aration layer, i.e., that the separation layer is meristematic. Hoehnel (1880) finds that cell divisions are characteristic of the proximal por- tion of the separation zone in Salix and Populus but in the distal portion, where the separation layer is developed, these divisions are not so numerous. In some cases he finds separation taking place in the parenchyma, entirely outside the "zone" where there had been no cell divisions. The separation cells in Nicotiana are described by Becquerel (1907) as small, practically undifferentiated cells with large nuclei. In Begonia, Fuschia, Mirabilis, and Impatiens Hannig (1913) describes the tissue as secondary meristem (type b) with the cells rectangular in shape and arranged in more or less definite rows. In contrast to the above observations, he describes the cells as small, irregularly arranged and spherical in Salvia, Solanum nigrum, and Nicotiana Tabacum. In Solanum nigrum the separation layer consists

1918] Kendall: Abscission of Flowers and Fruits in Solanaceae 353

of two or three tiers of cells but in N. Tabacum the layer is made up of ten to fifteen tiers.

Hannig (1913), by means of various microchemical tests, can detect no chemical difference between the cell walls of the separation layer and those of the cells on either side. Lloyd (1914a), however, claims that the cell walls of the separation cells break down more quickly when treated with caustic potash than do the walls of normal cells. Starch grains are frequently noted by Hannig and Lloyd (1916a) as occurring in the separation cells, especially in the abscis- sion of internodes by Mirabilis Jalapa.

An examination of the literature thus makes it evident that there has been a great difference noted in the various species in regard to the character of the separation cells. The one characteristic of these cells, however, to which there is no exception is that they are in an actively living condition.

2. THE PROCESS OF ABSCISSION

a. METHODS OF ABSCISSION

It has been found that in practically all cases of abscission the detaching of the organ is brought about by the separation of cells along the plane of the middle lamella. It is the method noted by Mohl (1860), Wiesner (1871), and Kubart (1906), who call it a pro- cess of maceration. Correns (1899) calls it a process of ' ' schizolysis. " Correns, however, in the same work describes a new and different method of abscission (rhexolysis) which he finds in mosses. In this latter method, separation is accomplished by a seemingly passive break of tissues irrespective of the position of cell walls. This may be the case in the style of cotton (cf. Lloyd, 1914&). This same method has been reported by Tison (1900) in the leaf of Aristolochia Sipho, although the evidence has been called in question by Lloyd and Loewi (1907). Still another type of abscission has been described by Hannig (1913) as a result of experiments on Mirabilis and Oxy- baphus. In these plants he finds separation being brought about by a disorganization and dissolving away of a complete tissue. Lloyd (1916a), on the other hand, states that separation in these species is accomplished by cell separation and is thus true schizolysis. Hannig was doubtless confused in this case by the cell elongations which Lloyd observes and by which the membranes surrounding the proto- plasts are drawn out exceedingly thin. Loewi (1907), working on

354 University of California Publications in Botany [VOL. 5

several genera, including Cinnamomum and Euonymus, notes and figures cell elongations similar to those figured by Lloyd (1916a). These cell elongations he finds so frequent and conspicuous that he proposes a distinct type of abscission, calling it a " Schlauchzell raechanismus. ' '

Loewi, on the basis of his studies, seeks to classify the methods of cell separation in abscission under six different headings, which per- haps would be more appropriately presented under the next subject of consideration (the methods of cell separation) ; but since the author gave them as distinct methods of abscission they will be considered here. They are: (1) "round cell" mechanism; (2) dissolving of the middle lamella; (3) maceration; (4) turgescence; (5) cell elonga- tions; (6) "hard cell" mechanism. They are to be considered merely as factors which, singly or in combinations, may enter in as a part of the normal process of cell separation. Loewi also claims that by con- trolling the temperature, humidity, and various other factors sur- rounding the plant he can influence it to such an extent as to change its method of cell separation.

ft. METHOD OF CELL SEPARATION

It has been held by various investigators that the cell separation, almost universally connected with abscission, can be caused either by (a) chemical alteration and dissolving of the middle lamella or by (&) increase in cell turgor. This whole matter has received consider- able attention, although very little direct evidence has been obtained. Wiesner (1871 and 1905) states that cell separation is caused by the dissolution of the middle lamella and by increased turgor. Kubart (1906) and Loewi (1907) agree entirely with Wiesner on this point. Strasburger (1913), Tison (1900), Lee (1911), Hannig (1913), and Lloyd (1916# and b) believe that cell separation is accomplished by the dissolution of the middle lamella. Practically all investigators have noticed the turgid appearance of the cells after separation, although this of course does not constitute evidence that the separa- tion is due to increased turgor. Fitting (1911) claims that the sep- aration is accomplished, at least in some cases, solely by an increased turgor of the separation cells. He bases his claim on the fact that abscission is very often too rapid to allow time for the dissolution of the middle lamella. He also mentions the fact that the separation cells are very often small, spherical cells, the type of cell which would respond most readily by an increase in cell turgor. On account of its

1918] Kendall: Abscission of Flowers and Fruits in Solanac&ae 355

rapidity and regularity of reaction, Fitting claims that abscission is a semi-tropistic phenomenon and suggests the term "Chorismus" to designate this type of reaction.

It has been observed by Hannig and Fitting that the presence of various narcotic vapors in the atmosphere around certain species of plants causes their flowers or merely the petals to be thrown off. Various aspects of this general problem of the reaction ofplant tissues to such agencies have been investigated. It has been determined by various plant physiologists that the presence of narcotic vapors, such as illuminating or acetylene gas, in the air around certain plant tissues causes the proportion of soluble carbohydrates within their cells to increase. This increase in the amount of soluble carbohydrates would indicate an increase in cell turgor. The question at once arises, whether or not this increase in turgor can effect complete separation or maceration of cells without the occurrence of chemical alteration in the walls. Kichter (1908) resting his case on experimental evidence, throws some light on this problem. Various kinds of plant tissues which he subjected to acetylene vapors broke in pieces because of the maceration and collapse of the living cells within. He finds that in the case of the cells of tissues which are commonly rich in starch inclusions, such as the fruit of the snowberry and the potato tuber, the maceration is most complete. In the potato, for example, 3 to 5 mm. of material on the surface become completely macerated after being subjected to acetylene gas. According to Bichter and Grafe (1911), the proportion of sugar in starchy seedlings subjected to acetylene gas is larger than in seedlings grown under normal condi- tions. In seedlings from oily seeds, however, the amount of sugar is decreased and the proportion of glycerine and fatty acids increased. The conclusion is therefore drawn that the subjection of plant tissues to narcotic vapors favors the hydrolysing process in the cells involved. The work of these two investigators goes to show that narcotic vapors may cause abscission by acting in either of the most important meth- ods suggested as responsible for cell separation ; they may increase cell turgor on the one hand or favor the hydrolysis of the middle lamella on the other.

Lloyd (1916a) presents evidence of chemical change in the cell walls of the separation layer before abscission. These cell walls stain in the usual manner with iodine, giving a light brownish color, but as abscission commences, they give a faint blue color when stained with iodine and washed out with water. Shortly before cell separa-

356 University of California Publications in Botany [VOL. 5

tion commences, Bisrnark brown and Ruthenium red fail to stain the primary and secondary cellulose membranes of the separation cells, although, when abscission does not occur, the entire cell wall is stained in the normal manner. The cells when separating seem, furthermore, to be surrounded only by the thin tertiary membranes. Lloyd, in his work, figures cells in the process of separation which show the disso- lution of the primary and secondary membranes of the cell wall.

Various interpretations are given to the repeatedly observed occurrence of cell divisions preceding and accompanying abscission Mohl (1860) expresses the opinion that cell divisions are generally necessary before abscission can occur. Investigators since his time have disproved the universal occurrence of cell divisions because they find more and more cases where no cell divisions occur. Lloyd (1914a) maintains that cell divisions are not of necessity correlated with abscission but are merely evidences of renewed growth and wound responses. As evidence he states that cell divisions are some- times absent and sometimes present in the same species. He cites (19165) the cotton plant as a typical example in which cell divisions are present in the abscission of older flowers in which the reaction to stimulus is slow. In young flowers and flower buds abscission may proceed without cell division. He further notes (19140) that cell divisions sometimes precede and at other times follow abscission in a given species.

c. AGENCIES ACTIVE IN BEINGING ABOUT THE DISSOLUTION OP THE MIDDLE LAMELLA

Very few theories have been proposed to account for the dissolu- tion of the middle lamella and practically no evidence of any kind has been submitted. "Wiesner (1905) claims that in leaf -fall an organic acid, produced as a result of lessening of cell activity and stagnation of cell contents, acts on the middle lamella. His evidence for this statement has to do with obtaining acid reactions with litmus from cells at the base of the petiole during abscission. Kubart (1906) also obtains acid reactions at the base of the corolla in Nicotiana dur- ing abscission and, although agreeing with Wiesner that an organic acid probably causes the dissolution of the middle lamella, he also admits the possibility that an enzyme plays a part in the process. Lloyd (1916&) makes the statement that the dissolution of the middle lamella is a process of hydrolysis and although making no definite statement on the subject appears to take it for granted that an

1918] Kendall: Abscission of Flowers and Fruits in Solanaceae 357

enzyme of some kind is the active factor. Indeed, since all hydrolys- ing processes of living cells are now supposed to be due* to the action of enzymes, there is no reason to suppose that the hydrolysis of the middle lamella does not conform to the general rule. For it is known that an enzyme, pectosinase, is capable of breaking down the pectose of which the middle lamella is composed. However, until more is known concerning the nature of this particular enzyme ~it remains impossible to get more definite evidence on this phase of the problem.

3. ABSCISSION OF THE COROLLA

Reiche (1885) gives an account of the fall of the corolla in a large number of species belonging to about forty-five families of the monocotyledons and dicotyledons. He finds that the corolla may be thrown off in one of three different ways: (1) by the activity of a small-celled separation layer; (2) through decay; (3) through in- crease in size of the ovary, thus tearing off the tissue involved at the base of the corolla. In many cases of true abscission — case 1 above — Reiche finds that the separation layer is preformed and ready to function at any moment. This represents a contradiction of Mohl's observations, according to which the fall of the corolla is usually due to the action of a separation layer formed shortly before fall. According to Reiche, the separation layer is very seldom morpho- logically differentiated from the neighboring tissue, but in a few cases he describes the separation layer as consisting of a layer of cells smaller than the neighboring cells on either side.

Kubart (1906), in his account of abscission of the corolla in sev- eral different species, describes and figures the process which takes place in Nicotiana. The separation layer in this genus he finds to be in no way morphologically differentiated, of indefinite shape, and located about 1 mm. above the base of the corolla tube. In this gen- eral region a large number of cells separate from one another, all the cells in cross-section taking part except the epidermal cells and the tracheae. Fitting (1911), in his work on the shedding of petals, de- scribes the process of abscission in several genera, paying particular attention to Erodium, Geranium, Linum, Helianthemum, Perlagonium, and Verbascum. Separation in these cases takes place through a region of small, spherical cells rich in protoplasm. The separation layer is not sharply differentiated as compared with the tissues on either side but is located in a restricted region at the base of the petal.

358 University of California Publications in Botany [VOL. 5

He finds no cell divisions preceding or accompanying abscission. The process in premature abscission he finds differing in no way from that in normal abscission after fertilization. These conditions, he states, correspond more or less to those which he finds in the pedicel during flower-fall.

4. TIME OF ABSCISSION

The time elapsing between anthesis and flower-fall in partially sterile F1 species hybrids of Nicotiana and between emasculation at anthesis and fall in the case of their corresponding parents is dis- cussed in a previous paper (Goodspeed and Kendall, 1916). It was there stated that the average time is about nineteen days in Fl H154, seven in Fx H179, five in N. Tabacum var. macrophylla, and thirteen in N. sylvestris. When we turn to the question of the reaction time in premature abscission occurring before the normal time as the result of sudden changes in external environmental conditions, we find that this subject has received only slight attention. According to Lloyd (1914a), the cotton "square" falls in one to twenty-two days after the weevil lays its eggs, the average time being eight days. In one experiment in which the ovary was cut transversely, Lloyd was able to cause one hundred per cent of the young bolls to fall in forty-eight hours and ninety per cent in twenty-four hours. Larger bolls take a longer time to respond to injury than do smaller ones, as a result of the development of the pedicel to a condition in which abscission meets greater resistance. Cotton * ' squares, ' ' he finds, take a longer time to respond than young bolls, the former shedding thirty-five to sixty per cent in thirty-six hours and the latter forty to seventy per cent in forty-eight hours. On the other hand, he obtains no evidence (19166) that the reaction times are any shorter in small buds than in larger ones. The reaction times in cases where the injury is performed in the evening seem to be shorter by about twelve hours than in cases where the injury is performed in the morning. This difference he ascribes to the increase in turgidity which takes place during the night and which serves to hasten the reaction. Very severe injuries to the ovary, he finds, cause fall of young bolls quicker than less severe injuries. Injuries which are less severe than those mentioned above and per- formed so as to imitate the injury inflicted on the ovary by insect larvae caused shedding in three to six days, with most of the fall occurring on the fifth day. Summing up his entire results, Lloyd

1918] Kendall: Abscission of Flowers and Fruits in Solanaceae 359

(1916&) states that under field conditions the responses to all kinds of stimuli conducive to abscission become evident within ten days, with the maximum frequency below six days.

The actual time involved in the process of abscission (abscission time) has received even less attention than the problems discussed above. Fitting (1911) states that abscission time may occasionally be very short, forty-five seconds to five minutes in the petals of Ver- bascum and thirty seconds to six minutes in Geranium. Lloyd (1914a and 19166) finds abscission after injury of the small cotton-boll taking place within four hours, the length of time depending somewhat on the age of the boll. In a previous paper (Goodspeed and Kendall, 1916) a general estimate of the abscission time was given and it was stated that normal abscission due to lack of fertilization takes place in Nicotiana hybrids in four to eight hours and premature abscission in one to four hours.

5. EXPERIMENTAL INDUCTION OF ABSCISSION

According to Hannig and Loewi, abscission may be induced in two different ways. First by abnormal external conditions ("spon- taneous" or premature abscission) and second by normal internal conditions at the normal time ("automatic" or normal abscission). We shall consider in the following summary of the literature only two aspects of induction of the first type.

a, INDUCTION BY NAECOTIC VAPOES

Hannig (1913) reports a comparative study of the behavior of cut sprigs of different species of plants when subjected to laboratory air and to illuminating gas. He notes the fact that under either of the above conditions all the flowers and occasionally a few small shoots are abscissed. He finds, however, that not all the species in a given family behave similarly in response to these conditions. We are particularly interested in the Solanaceae and we may note that this family contained more species that detached their flowers in illuminating gas than any other of the families investigated by Han- nig. According to Fitting (1911), narcotic vapors such as tobacco smoke, carbon dioxide, ether, chloroform or illuminating gas fre- quently cause premature abscission of the corolla. He notices, how- ever, that ammonia or turpentine vapors fail to cause abscission. Brown and Escomb (1902) make the statement that Nicotiana, Cu- curbita, and Fuchsia shed flowers and buds in air containing only 0.114 per cent carbon dioxide.

360 University of California Publications in Botany [V<>L-

fc. INDUCTION BY MECHANICAL INJUEY

Becquerel (1907), in a brief paper on the effect of wounding flowers of Nicotiana, notes that even after fifteen days flowers without sepals, anthers, or stigmas do not fall. After the same length of time, flowers without corollas or flowers in which the corolla or stamens are only half removed, have fallen. He points out that this result is more conspicuous in young flowers but did not investigate this point suffi- ciently to arrive at any definite conclusions. According to Hannig, removal of various organs of flowers frequently causes abscission but wounding of the pedicel does not. He concludes, therefore, that in- jury itself does not cause abscission but only acts indirectly by inter- fering with important physiological processes in the treated tissues.

According to Lloyd (1914a), shedding of very young cotton-bolls can be induced by removal of the styles before pollination, but fall in this case can be assigned, as Fitting has shown, to lack of fertilization. It appears that in the cotton flower (Lloyd, 1916&) there is an inhibi- tion period which starts with the opening of the corolla and during which premature abscission as the result of sudden stimuli very sel- dom occurs. Also, cotton-bolls larger than 30 mm. in diameter are very seldom shed under any conditions. Other results obtained by Lloyd on the effect of injury on the abscission of cotton flowers are discussed above under "Time of Abscission" (page 357). Lloyd (1914&) also notes the effect of injury on abscission of internodes in Impatiens Sultani. Plants of this species, when a cut is made across the stem, cast off the remainder of the severed internode. He gives results of experiments on the effect of different types of injury, noting that some severe injuries do not cause abscission. Gortner and Harris (1914) have obtained similar results with the same species. They find that when the cut is made across the internode, very close to the separation layer, abscission usually occurs, but occasionally it does not. They state, as does Lloyd, that the shape and location of the separation layer may vary slightly according to the type of injury.

c. THE DIEECT OE INDIEECT ACTION OF THE EXTEENAL STIMULUS

In all the above investigations the question naturally arises, whether the narcotic vapors and injuries or any stimulus conducive to abscission act indirectly through their influence on the physiolog- ical condition of the plant or directly, through their action on the cells of the separation zone. Most investigators, except Wiesner, ex-

19181 Kendall: Abscission of Flowers and Fruits in Solanaceae 361

press the opinion that atmospheric factors work directly in causing "spontaneous" abscission, although offering, so far as I can see, no evidence for this view. Fitting states that the external influence acts directly in most cases, but that the indirect action is apparent in forms which must build a separation layer before fall can occur. In regard to the action of injury, it seems to be the opinion of most investigators (Hannig, Bacquerel, Gortner and Harris)-- -that the stimulus acts indirectly by interfering in some way with such important physiological processes as transpiration, respiration, or assimilation. On the other hand, if abscission is sometimes a semi- tropistic phenomenon, as Fitting has suggested, it is evident that injury may act directly in causing flower-fall.

TECHNIQUE

The results noted below were obtained largely from the examina- tion of microscopic preparations made by the paraffin method, although this method was supplemented by free-hand sections mounted in water. In investigating the condition of the pedicel in some species (Datura sp., Petunia sp. and several species of Nicotiana) only free- hand sections were examined. For most microchemical studies fairly thick, free-hand sections are preferable. The material for sectioning in paraffin was killed and fixed in various concentrations of the chromo-acetic series and dehydration and infiltration were, in general, carried on very slowly. The free-hand sections were mounted in water without killing.

In cutting longitudinal sections of any kind all the pedicels were oriented so that the sections were cut parallel to the main stem of the inflorescence, in the plane formed by the pedicel and stem taken together. In studying the- histology of the pedicel and the cytology of the separation layer and in studjdng the method of cell separation, these longitudinal sections were supplemented by cross sections in series through the base of the pedicel. It was impossible to cut very thin, longitudinal sections in paraffin without crushing or breaking the cells ; most of these sections therefore were cut from 10/x to 15/* in thickness. For a similar reason, it was found necessary to cut thick sections (20/x to 25/*) of the pedicels of fruits in which mechanical tissue had developed. It was possible, however, to cut excellent paraffin sections from 5/x, to 1^ in thickness in cross-section or longitudinally through the small cells of the separation zone. Since the cells of the

362 University of California Publications in Botany [VOL. 5

separation zone are very small, not much could be determined in regard to the dissolution of cell walls by means of thick, free-hand sections. The best results along this line were obtained from the thin paraffin sections of the separation zone, although in order to show the cell wall in its normal thickness it was necessary to use the free-hand sections. As a supplement to these sections, several points of interest were brought out by washing off the isolated cells from the end of freshly abscissed pedicels and mounting them for microscopic exam- in.ation.

In most of the work the paraffin sections were stained in safraniii and Delafield's haematoxylin. The free-hand sections were generally mounted in water and stained in iodine. In special instances other stains were used. Thus, in testing for chemical differences in the cell walls of the separation cells, several other stains, such as erythrosin, eosin, Bismark brown, gentian violet and Ruthenium red were used. It was found that for demonstrating the dissolution of cell walls aqueous methylene blue was an excellent stain to use. This stain was allowed to act overnight and the sections destained slightly in alcohol. Methylene blue was also an excellent stain for the isolated cells ob- tained as noted above. By fixing these cells to the slide with albumen fixative and staining with this stain, the thin membranous wall sur- rounding the protoplast can be distinctly seen.

Various methods, such as subjecting inflorescences to illuminating gas and mechanical injury, were used to bring about abscission. The best results were obtained in cases where abscission was induced by inserting shoots under a bell-jar containing from 1.5 per cent to 3 per cent illuminating gas. By using illuminating gas in this way and by taking sections of the pedicels at intervals it was possible to determine just when the first signs of abscission appeared in a certain percentage of gas. This time was definitely determined for certain species so that it was possible to get material killed and fixed at any desired stage in the process of abscission. It was found that the best results were obtained by killing and fixing the pedicels at about the time when abscission was known to be commencing.

1918] Kendall: Abscission of Flowers and Fruits in Solanaceae 363

HISTOLOGY AND CYTOLOGY OF THE PEDICEL

1. HlSTOLOGICAL AND CYTOLOGICAL CONDITIONS OF THE

MATURE PEDICEL a. NICOTIANA

The vascular system in Nicotiana, as in all the other genera examined, is characterized by intraxylary phloem. Nicotiana differs slightly from all others in that the xylem seems in cross-section to be composed of a continuous ring of radial strands of tracheae rather than composed of a broken ring of distinct bundles. When a branch of the vascular system (fig. 1, a) containing twenty to thirty xylem strands is given off to the pedicel, it assumes the shape of a crescent in cross-section, with the opening of the crescent on