Vernalization and Photoperiodism, p. 94 (1948)
Anatomical and Histological Changes in Relation to Vernalization and Photoperiodism
R. H. ROBERTS and B. ESTHER STRUCKMEYER
University of Wisconsin

The cause of flowering in plants has been the subject of a rapidly increasing amount of literature in the past few decades. The majority of such papers have dealt with the environmental factors employed to induce blossoming rather than with any possible basic physiology of sexual reproduction. That is, such items of external environment as the degrees of temperature and quality or duration of the daily light have been more often the subject for investigation and emphasis than have the conditions which might consistently occur within the plant when blossom primordia are initiated.

The opposite effects which comparable applications of nitrogen have upon the blossoming and fruiting of apple trees which are in widely different stages of vigor directed attention toward the possibility of using histological conditions and anatomical characters as an index of the beginning of reproductive processes (16, 21). Observation of the photoperiodic effects and particularly the influence of temperature upon photoperiodism (27, 28, 30) increased interest in the question: Are there common physiological conditions present within plants and also common physiological reactions by different species of plants whenever sexual reproduction is initiated, even though uses of different temperature, light, water or nitrogen nutrient conditions are needed to bring about flowering?

Four different lines of evidence have been secured which indicate a common sequence of physiological events when blossom induction occurs: (1) The rhythm of CO2 exchange was found to be unlike in flowering and non-flowering plants (24, 25). This was true of both long- and short-day plants. (2) The daily growth rate and diurnal cycle of growth change very quickly when plants are placed in conditions leading to blossom induction (11). (3) Root extension is greatly reduced when plants enter the reproductive state. That is, top-root ratios of a number of plants were found to be related to flowering of the plants and not to the day length used to induce blossoming (32). (4) A large number of varieties of plants, including day-neutral and long- and short-day types, and those with high or low temperature preference exhibited common anatomical characters when placed in an environment inducive to blossoming (32, 33, 36). A review and extension of these latter observations, together with a consideration of the available references to similar work and their significance to photoperiod and vernalization is the subject of this paper. A fifth physiological condition has recently been investigated (23). This is pigment content. It has been found that the fractions of chlorophyll, carotene, violaxanthin and xanthophyll present during the daytime as measured by the chromatographic method do not appear to be related to blossoming. Also, pigment conditions during the dark period are not consistently correlated to reproduction. It appears that the diurnal cycle of pigment content, especially carotene, varies with day length rather than with reproductive state.

Experimental Evidence:—Interest in the possibility of the anatomical structure of plant stems being associated with flowering arose from the [92] observations that sour cherry (Prunus cerasus) (17), plum (Prunus nigra Hort.) (19) and apple (Malus malus) (20, 21) have a greater stem diameter when blossom buds are formed. Later observations (38) on the time of apple blossom bud induction disclosed that the marked secondary thickening which is typical of "fruit" spurs is produced after blossom bud initiation has begun. The anatomical differences which are characteristic of non-flowering and flowering stems of the apple are like those previously found in annual and biennial plants (33, 37) : "The flowering stems of all the species examined (at the fourth internode from the stem tip) seem to have certain anatomical characteristics in common, regardless of age or of photo periodic classification. In contrast to the non-flowering stern, the flowering stem is characterized by: (1) a less active cambium; (2) a zone consisting mainly of thick-walled secondary xylem elements lying adjacent to the cambium in contrast to rather numerous vessels and thin-walled parenchymatous cells in the last formed xylem of the non-flowering stems; (3) generally thicker walls of the cells of the pericycle, perimedullary zone, and phloem; (4) freer staining with 'basic' dyes of the pericycle, perimedullary zone, and certain elements of the xylem."

Typical changes in the structure of the phloem as the reproductive state is entered were also found (33) : "Some of the phloem characteristics which have been seen to accompany blossoming are: (1) Limited or slight formation of phloem cells following reduced cambial activity which precedes blossoming; (2) Small size of later formed cells; (3) Increase in cell wall thickness; (4) Increase in callose formation on sieve plates and fields; (5) Accumulation of inclusions in some cells; (6) Mechanical com pression."

Additional observations upon the relation of cambial activity to the reproductive condition of some dicotyledonous plants (39) led to the following summary statements: "Vigorously vegetative plants have an active cambium throughout the length of their stems. The cessation or decrease of cambial activity which accompanies the production of flowers progresses from the region of the inflorescence toward the base of the plant, which it may or may not reach depending upon the degree of reproductiveness which the plant attains as measured by the relative number of primordia which differentiate as floral structures. If certain species of plants are allowed to reach an advanced stage of reproductiveness under favorable environmental conditions, the meristematic tissue of their stems tends to become entirely differentiated into xylem and phloem elements. This anatomical condition is a possible explanation of the death of such plants at the close of one reproductive cycle." WILTON had ample evidence to have proposed that the annual habit is due to a cession of cambial activity rather than to have only suggested this possibility.

1 PAUL BERNSTEIN, LOIS THOMSON FOSTER, DR. B. E. STRUCKMEYER.

Unpublished work upon the cambial condition in roots1 has shown that the cambium becomes inactive or "lost" in maturing annual plants and fruiting biennials and that it persists in perennials and in those annual or biennial plants which have been kept in an environment which maintained them in a non-flowering state.

The relation of cambial activity and sexual reproduction has been observed in all of 65 to 70 species of dicotyledons which have been examined.

The time and nature of the transition from the anatomical condition which is typical of non-flowering plants to that found in the flowering stems has been determined and described (34). A marked reduction in cambial activity and a corresponding increase in maturation of xylem and phloem elements was clearly apparent in stems of Salvia splendens var. Harbinger in 5 days after placing the plants in an environment inducive to flowering (short days and a warm night temperature). This is three to four days prior to the appearance of blossom primordia.

2 From cultural comparisons and reactions to photoperiod
this is quite obviously the same species as that used by HAMNER
and BONNER (7) and called X. pennsylvanicum by them.

Different species exhibit different rates of reaction to an environment favorable to blossom induction. At optimum photoperiod and temperature, soybeans (Glycine max var. Biloxi, a short-day type) showed marked anatomical changes in five to six days and blossom primordia in nine to twelve days. Xanthium echinatum2 responds more rapidly; anatomical changes were obvious in three days and blossom primordia were found in four to five days after the start of short photoperiod treatments. Cosmos sulphureus var. Kiondike showed very distinct changes in anatomical structure after three short days and blossom primordia were observed in twelve days.

In the long-day species Matthiola incana (Stock, var. Christmas Pink) blossom primordia were found after 17 to 18 long light periods. Changes in anatomical characters were found in these plants after only five to six days of treatment.

From the preceding paragraphs it is evident that the first appearance of blossom primordia would not be an index of when induction begins. In fact, in the apple (38) and cranberry (31) induction has progressed to a stage where nearly complete defoliation will not interrupt it, as long as three to four weeks before primordia arise. This would seem to bring into question the desirability of dissecting the tips of plants to observe the appearance of primordia as is insisted upon by some workers. Changes in anatomical structure appear to correspond very closely to induction. Observation of this situation may be found to be useful if it is desired that the time of initiation be determined accurately.

The length of photoperiodic treatment needed sufficiently to induce a plant so that it becomes sexually reproductive even after being transferred to an environment inhibitory to induction also varies with the species or variety. Biloxi soybeans need approximately 17 short days (34) to establish effective "after-affects." It will be reported later that plants which flower terminally as Salvia splendens var. Harbinger will return to a vegetative type of growth even well after they are in flower if the plants are given long-day conditions. That is, they do not exhibit fixed after-affects. It is reported (7) that Xanthium becomes sexually reproductive after only one long dark period (one short day) even though returned and continued in long days. Staminate flowers will usually but not always be produced by plants given one long dark period but in repeated trials it has [94] been found that two or more long nights are necessary for the subsequent development of pistillate flowers.

New Evidence:— Changes in anatomical structure have appeared consistently with or prior to blossom induction when usual conditions are used. They have also been well in advance of blossom primordial. There remains the question of the anatomical history when blossoms are initiated under such unusual environmental treatments as reversed night temperatures (22), "temperature girdles," banding, and grafting (29).

When plants which come to flower early under relatively warm night temperatures as, Cannabis sativa (hemp), Datura stramonium (Jimpson weed), Euphorbia pulcherrima (poinsettia), Glycine max (soybean, var. Biloxi), Nicotiana tabacum (tobacco var. Maryland Mammoth), Panicum milaceum (millet), Phaseolus vulgaris (kidney bean), Solanum capicastri (ornamental pepper), Xanthium echinatum (cocklebur) and Zea mays (corn) are transferred to an environment with a warm (75°F) dark period of 13 hours and cool light period (55°F) of 11 hours, they soon become nearly etiolated or at least produce new growths with very little green color (23). Such plants are of particular interest from the standpoint of the physiology of sexual reproduction. They differentiate and develop blossoms and may even set fruits at almost the same rates as normally green plants in cool nights and warm days. Long continued development is not usual with such pale plants but the initiation of sexual reproduction is not inhibited and only slightly delayed by a lack of green color.

Examination of the stems of plants grown with warm nights and cool days discloses that they have an anatomical history parallel to those grown in cool night and warm day temperatures. While they have the thin-walled cells with slight inclusions which are typical of partly etiolated or shade-grown samples, reduced cambial activity preceded the appearance of primordia when the proper photoperiod was used to induce reproduction (Figs. 1-4.).

FIGURES 1-4.—Nicotiana tabacum VAR. Maryland Mammoth: 1, Non-flowering plant in long photoperiods with cool nights and warm days. 2, Flowering plant in short photoperiods with cool nights and warm days. 3, Non-flowering plant in long photoperiods with warm nights and cool days. 4, Flowering plant in short photoperiods with warm nights and cool days. Cessation of cambial activity precedes blossoming.

Three unusual methods of inducing blossoming also resulted in plants with characteristic changes in cambial and cellular condition prior to and during blossom bud development. The short-day plants, poinsettia (Euphorbia puicherrima) and Maryland Mammoth tobacco (Nicotiana tabacum) do not blossom in short days at warm night temperatures of 75°F (30). They will blossom in this environment if a current of cool air is passed through a short chamber placed about the stems some 3 to 4 nodes below the tips (29). The structure of the stems above the "temperature girdles" becomes typical of those induced to blossom by usual induction techniques (Figs. 5, 6). BORTHWICK, PARKER and HEINZE (2) report that cooling of the petiole of the leaf responsible for providing the flowering stimulus prevented the transfer of the stimulus and inhibited blossoming of Biloxi soybean.

Poinsettia plants in short days, but too warm at night to initiate blossom, can also be brought into blossom by wrapping taut rubber bands about the stems near the tips. When blossoming is induced in this manner, characteristic changes in the anatomical structure were found to occur above the point of banding.

FIGURES 5, 6.—CROSS-SECTION OF ITEMS OF POINSETTIA (Euphorbia pulcherrima): 5, Non-flowering stem. 6, Flowering stem sampled above a "temperature girdle" has a cambial condition typical of flowering stems.—FIGURES 7, 8.—Spodograms of Xanthium: 7, Ash residue of vegetative stem. 8, Same after five short photoperiods.

[95]

3 It is suggested that inexperienced workers take care to use correct temperatures
when experimenting with Hyoscyamus niger as this plant appears more responsive
to different levels of temperature than to long or short photoperiods.

The induction of sexual reproduction by grafting a flowering plant upon a "receptor" plant has been reported by a number of workers (33, 7, 8, 10, 41). When plants are brought to flower by the grafting technique the receptor plants exhibit anatomical changes similar to those induced to blossom through photoperiodic conditions, temperature or age.

Two other lines of evidence show a correlation of anatomical structure with sexual reproduction. One is the characteristic amount of phloem tissue in a species and the other is the renewal of cambial activity when plants which are in flower are made to produce new vegetative shoots (34). Its significance is not known but it is very suggestive of a common physiological basis for sexual reproduction that several species of plants which blossom early and continuously in a very wide range of environmental conditions as buckwheat (Fagopyrum esculentum) were found to have a very limited amount of phloem tissue (37) ; also, that other species which can rarely be induced to form blossoms as commercial varieties of sweet potato (Ipomoea batatas) had a characteristically abundant formation of phloem.

The renewal of cambial activity after the induction period as in cases like the unilateral production of xylem cells resulting in the large-diameter spurs characteristic of the apple when blossom buds are developing, the growth of perennials after a rest period or the return to active cambial formation when plants are replaced in an environment unfavorable to blossom initiation (34) will not be considered now. Neither will a discussion of the questions arising in connection with late bud development, flowering, fruit set and fruiting be undertaken. Conditions most conducive to induction are of course often very different from those giving optimum fruiting in a commercial sense.

Literature:—There are rather numerous references to the effects of photoperiod or other external environmental factors upon stem anat omy (9). As the plants have been sampled according to the cultural treatment and not the reproductive state, it is not usually known but can only be guessed as to whether the anatomical condition observed bore a relation to blossom initiation. An example is HAMNER'S statement (6, p. 586): ". . . . PSAREV et al. found an increased cambial activity during induction."

The papers by PSAREV (12, 13) and PSAREV and NEUMAN (14) which HAMNER cited do not record the cambial activity nor do they describe the cellular structure either during induction or at a later time. They report only an increase in stem diameter of mature soybean plants grown in short days (presumably due to longer continued cambial activity.) The short day plants of PSAREV (14) had some internodes which "assume a shape of abnormal swellings or 'tumors' which look like those induced by several different authors through treating the plants with growth substances." From this fact and also the statement that mature plants in short days averaged only 10.2 and 15.6 cm. in height, it appears that PSAREV was working with abnormal plants such as occur in short days in the greenhouse when the [96] nights are cold (55°F) and which also appear in the fields of the Midwestern United States of America as what farmers call "duds" (36).

A detailed study of the anatomy of this type of plant shows definitely that the large diameter is due to increase in parenchyma cell size and not to increased xylem formation from cambial activity (Fig. 9). The thick ness of these stems and presumably also of the short day plants of PSAREV, is due to a combination of low temperature and short days and is not associated with induction. Plants which fruit normally in short days and warm night temperatures remain slender. Also, they have the anatomical conditions typical of reproductive plants (Figs. 10, 11). More evidence is needed on the relation of anatomical condition and reproduction. This should be secured by sampling and studying plants at the time of induction and early blossoming in comparison with actual non-flowering plants.

FIGURES 9-11.—CROSS-SECTIONS OF STEMS OF BILOXI SOYBEAN: 9, Abnormally large and distorted parenchyma cells of "dud" plant grown in cool, short photoperiods (Magnification .52 times that of figures 10 and 11). 10, Non-flowering stem of young plant before induction. 11, Flowering stem of plant grown in warm, short photoperiods.

WITHROW (40) has found an anatomical condition in non-flowering and flowering plants like that described by WILTON and by STRUCKMEYER. This consisted of reduced cambial activity and limited phloem formation in plants which flowered. This was found to be true of both long- or short day plants and also occurred without regard to the nitrogen nutrition.

Plant Differences:—In the course of the induction of blossoming by numerous methods to ascertain the consistency of anatomical history of sexual reproduction, a particularly interesting difference in the reaction of plant species has been observed. We do not refer to the different or even opposite flowering responses of different species to like photoperiods or temperature treatments or age but to another plant characteristic which appears to have as much effect upon the type of experimental evidence which is secured as does the influence of certain external conditions. We refer to the blossoming habit: Plants which have only terminal blossoms react differently from those which have blossoms at numerous growing points along the stem; i.e. those which flower systemically (29). Thus GARNER and ALLARD (5) showed there was no transfer of the flowering stimulus between branches of Klondyke cosmos (Cosmos suiphureus), a terminal flowering species. At much later dates morning glory (Ipomoea purpurea var. Heavenly Blue) (27), Xanthium (7), soybean (Glycine max) (1) and other plants with a systemic flowering habit were reported to show a transfer of the flowering stimulus.

This relation of terminal and systemic flowering habits to reaction to flowering stimulus from donor branches or leaves has been checked in only a few score of plants. It is anticipated that an exception to the general classification may be found. The nearest to it to date is Perilla frutescens (nankinensis). No transfer of the flowering stimulus with this systemic flowering plant has been secured in our trials even when the leaves are removed from the receptor branches. In the experience of some workers it is also necessary to keep the flowers removed from the donor portion. We have not tried this technique.

It is also interesting that plants flowering terminally are not induced to blossom by grafting the stem of a flowering plant onto a non-flowering one. (It would be interesting to know what the results of MOSHKOV (4, p. 230-1) would have been had he "decapitated" the receptor tobacco plants at a lower level, that is, in a region of the stem where blossoms do not ordinarily [97] arise.) Several species which produce blossoms at any node have been readily induced to flower by the grafting technique.

The blossoming habit of the plant should be considered when "transfer" experiments are being planned. It should also prevent unjust criticisms being made such as have begun to appear in the literature, when two workers obtain different results, obviously from the use of plants of different flowering habits.

After-Affect:—Another phenomenon which varies with the flowering habit of the species is the difference in reaction to a change in environment, after the plant has come to flower. Plants with a terminal flowering habit as Klondyke cosmos, poinsettia, Rudbeckia laciniata, Salvia (var. Harbinger), stock (Christmas Pink) and Maryland Mammoth tobacco can he readily changed to a vegetative state after they have come to flower by placing them in an environment which inhibits flowering, plants with a systemic flower forming habit as morning glory (var. Heavenly Blue), petunia (forcing), soybean (var. Biloxi), and Xanthium echinatum do not revert to a vegetative growth cycle in a reversed environment, once they have come to flower and fruit.

The continued flowering of an induced plant after being transferred to an environment unfavorable to flowering (provided it is of a species having a systemic blossoming habit) presents an interesting problem. The mechanism of the "after-affect” has been the subject of considerable theorizing. A possibility of an effective mechanism is presented by the fact that annual plants which show after-affects do not characteristically renew cambial activity once it has ceased at the time of induction and flowering (34). In this connection it must be kept in mind that plants which require a long period of treatment to establish a permanent after-affect will revert to a vegetative type and regenerate cambial activity if the induction treatment is discontinued before induction is completed. Terminally flowering varieties regenerate cambium whenever returned to an environment unfavorable to blossom induction.

Monocotyledons:—An extensive study of the relation of stem anatomy to flowering of monocotyledons has not been attempted as so few of the commonly available species have stems suitable for sampling when in a non flowering state. The grains and grasses as well as most other locally grown species of this group are in an induced state before stems long enough for sampling of internodes are produced.

Histology:—Too little original work has been done on the question of the relation of plant composition to blossom induction to be of much significance. It is of interest that the etiolated type of plants grown in warm nights and cool days may have so little carbohydrate that no starch and almost no free reducing substance is found by qualitative tests.

Consistent differences in the mineral pattern of flowering and non-flowering stems have been found (35). It has also been repeatedly observed that a change in the mineral distribution becomes apparent very soon after plants are placed in an environment inducive to flowering. A changed pattern is evident in as short a time as two to three days in some species (Figs. 7, 8, opposite p. 95).
[98]

Like most of the anatomical studies reported in the literature, analytical studies have been more commonly on samples from different environmental treatments or of plants in later stages of flowering or fruiting rather than of samples in early stages of sexual reproduction (9). To secure evidence on the physiology of sexual regeneration, sampling of potentially flowering plants should be done at the time of blossom induction. Similarly, cytological studies of the apical meristems involved in the differentiation of floral primordia are needed.

Role of Anatomy:—That no possible misunderstanding arise, it is clearly acknowledged that the significance of the anatomical situations which have been found to be associated with sexual reproduction is not known.

4 From our observations, a transfer of the stimulus-to-flower by grafts separated by a "diffusion contact" of lens paper (7) is not possible unless the proliferating tissue has penetrated the separating medium and made a direct contact between the donor and receptor stems as found by MOSHKOV (10) and by WITHROW and WITHROW (41). MOSHKOV reports a 10-day "physiological contact" is necessary to secure induction with Perilla grafts.

It is difficult, however, to avoid considering that the stopping of phloem formation at the time of induction and prior to blossom differentiation and flowering might not greatly affect transfer of the flowering stimulus as well as conduction of other elaborated substances from the leaves.4 On the other hand, the evidence is not now sufficiently definite to justify proposing that anatomical conditions are causative in sexual reproduction, even though the possibility seems to exist.

Phytohormones:—The phytohormones have been considered as causal in blossom initiation by numerous workers (chapter 6, this series). At least, names have been proposed ("florigen" of CAJLAHJAN and "anthesin" of CHOLODNY) for the as yet unextracted substance which is presumed to pass through a graft union and cause the flowering of a receptor plant. If phytohormones are the real stimulating agent they obviously work through the mechanism which inhibits cambial activity and induces maturation. This gives a clue as to the physiological reaction they would induce. A substance which would result in maturation phenomena would be un like any commonly known phytohormone as the ones now available for plant treatments induce proliferation. Should a "maturity inducer" be isolated it should not only be of potential use in causing flowering but also be a possible remedy for cancerous growths, that is, if cancer is "the inabil ity of cells to stop growing." Methods of extraction should be directed toward the securing of such a substance.

Relation with Photoperiodism:—Since anatomical conditions have been seen to be correlated with sexual reproduction, it is very obvious that a given photoperiod, as for example a short day, would not result in a common anatomical situation in different plants as it would either induce blossoming (of a short-day plant), inhibit blossoming (of a long-day plant), or have little if anything more than a time effect upon blossoming (of a day-neutral type).
[99]

Relation with Vernalization:—A comparable situation exists in connection with any possible relation of vernalization and anatomical de velopment. If vernalization be given a narrow definition, as a cold treatment, or if it be given a wide meaning, as any blossom-inducing treatment even including photoperiod as one item, it would not have a consistent relation with anatomy as no one factor of the external environment as moisture, nutrient, grafting, girdling, temperature, etc., have the same effect upon the inducing of sexual reproduction of different types of plants. Flowering is, however, correlated with anatomical development.

It would be interesting to know whether there are anatomical changes in the plants exhibiting devernalization comparable to those found in plants which do not have persisting after-affects (34).

Relation with Phasic Development:—The relation which has been found to be consistent between anatomical changes and the start of the cycle of sexual reproduction (blossom induction) should be a means by which to measure the general applicability of the proposition that the plant comes to flower by a series of phases, each of which must be completed before a succeeding one is entered.

Some interesting materials for study would be:

  1. Pigweed (Amaranthus retroflexus) seedlings which flower with the first pair of leaves and even prior to the differentiation of secondary tissues, when grown in warm, short days.
  2. The quick change from a non-flowering to a flowering state of Xanthium after two long-nights (staminate blossoms may be induced by only one long dark period).
  3. Reversion to the vegetative condition of terminally flowering types of plants following a change of environment. (Rather spectacular examples can be produced with such plants as beets (Beta vulgaris) as the stems thicken into "beets" when the plants are transferred to an environment unfavorable to flowering.)
  4. The continued flowering of plants with a systemic flowering habit after complete induction although having been moved into an environment unfavorable to induction.
  5. Plants like Nicandra physalodes and buckwheat (Fagopyrum esculentum) which start to flower under very wide extremes of environmental condition and continue to grow and initiate and develop flowers even after maturing fruits are present on the plants.

It is suggested that observation of the anatomical conditions in the plant might be an aid in determining the boundary between the vegetative and re productive phases. There is a relatively long lapse of time in many plants between the start of induction and the appearance of even microscopic blossom primordia. (Observation of the cambial condition in petioles can be made without greatly modifying a plant for later observation.)

Comment:— Use of the techniques of photoperiodic and vernalization experiments does not appear to offer solutions of the problem of sexual re production. The manipulation of external factors gives a record of the treatment needed to initiate sexual reproduction and thus serves as an aid in the control of plants for commercial or experimental purposes. An understanding of the physiological processes of reproduction would appear to depend upon a study of the internal factors and mechanisms involved [100] rather than upon an increase in knowledge of how to regulate the environment to induce flowering.

Anatomical changes in the stems (sampled at the fourth internode from the tip) have been observed to be consistently related to the entrance of a plant into the reproductive state. After-affects which are characteristic of some plants (with a systemic blossoming habit) and not typical of others (with a terminal blossoming habit) are associated with cambial activity. In fact, this difference in blossoming habits suggests a possible difference in the transport systems of different species.

A study of the relation of the anatomical development of a plant to sexual reproduction indicates that an item which is urgently needed for a solution of why plants blossom would be the extraction or discovery of a substance which would produce maturation phenomena when applied to or introduced into a non-blossoming plant, in contrast to the producing of proliferation phenomena by presently known phytohormones.

References:—