DEVELOPMENTAL ANATOMY OF THE STEM APEX
OF THE BETTER TIMES ROSE
DISSERTATION
Presented in Partial Fulfillment of the Requirements
for the Degree Doctor of Philosophy in the
Graduate School of the Ohio State
University
by
Richard Stadden Lindstrom, B. Sc,. ^ . M. Sc.
The Ohio State University
1956
Approved by:
Adviser
Department of Horticultui and Forestry ACKNOWLEDGEMENT
There are many to whom I owe acknowledgement for the completion of this dissertation. In particular, I wish to express sincere appreciation to Professor Alex Laurie for suggesting the problem and to Dr. L. C. Chadwick and to Dr. D. C. Kiplinger for making it possible to complete the work. I also wish to thank Dr. R. A. Popham,
Botany Department, for his constant interest and guidance on the anatomical aspects of the problem.
Appreciation is also extended to Roses Incorporated, whose fellowship I held for three years and to the Department of
Horticulture of Michigan State University for the use of their laboratory and the many hours of freedom for investigation.
P. G. Coleman's assistance with the microphotography and
Dr. R. F. Stinson's suggestions are also appreciated. TABLE OF CONTENTS
Page Introduction ...... 1
Review of Literature ...... 3
Materials and M e t h o d s ...... , 13 r
Results ...... , 19
Discussion ...... 43
Summary ...... 49
Bibliography ...... 50
Autobiography ...... • • • 52
iii LIST OF ILLUSTRATIONS
Figures Page 1. A vegetative stem apex from a shoot that presumably would have been flowering (terminal)...... 20 2. A vegetative stem apex from a shoot that presumably would have been blind (axillary)...... 21 3. The first evidence of reproduction in a presumably flower ing s h o o t ...... 23 4. An early stage of sepal primordia ...... 24 5. A later stage of development of sepal primordia ...... 25 6. An early stage of petal primordia which appeared 13 days after the cutback ' ’...... 26 7. A later stage of petal primordia formation ...... 27 8. An early stage of pistil primordia ...... 28 9. A later stage of pistil primordia development ...... 29 10. The first evidence of reproduction in a presumably blind s h o o t ...... 31 11. An early stage of sepal primordia in a presumably blind s h o o t ...... 32 12. The first evidence of disintegration of tissue in a presumably blind shoot ...... 33
13. Further disintegration of the s e p a l s ...... 34
14. Complete abortion of the rose apex at petal primordia stage 35 15. Intermediate stage of disintegration at stamen primordia s t a g e ...... 36 16. Abscission layer formed in the stamen primordia stage . . 37 17. Intermediate stage of disintegration at pistil primordia s t a g e ...... 33 18. A very late stage in the disintegration of tissue at the pistil primordia stage .... 39 19. An aborting rose apex showing necrotic bands and cell disintegration ...... 40 20. The formed abscission layer of an aborting a p e x ...... 41
iv DEVELOPMENTAL ANATOMY OF THE STEM APEX
OF THE BETTER TIMES ROSE
INTRODUCTION
Roses are the most important floricultural greenhouse crop in the United States. According to the Agriculture Census (1) the wholesale value of roses grown under glass in 1949 was $30,582,022.00 and for all cut flowers grown under glass was $93,478,944.00. A comparison of these values shows that roses constituted 32.7 per cent of the cut flower income for that year.
The greenhouse rose is an ornamental decidious shrub with upright or climbing stems which are usually associated with spines. The rose is classified under the family name Rosaceae. Accurate information on parentage and origin of the present day hybrid varieties is lacking but it has been reported by Wildon (21) that some of the species which are parents of the modern hybrid teas are R. gallica, R. damascena,
3i* centifolia, r . Borionica, and R. odorata.
Commercially the rose grower receives the greatest profit for his crop during the months of November through May. The midwestern growers have the most difficulty producing the crop during this season because
of low prevailing light intensities. The grower becomes increasingly
aware of the unproductiveness of "blind wood" (a shoot with an aborted
- 1 - - 2 - flower bud), which reduces his potential crop and therefore profit.
It would be advantageous if the cause could be found or the incidence of blind wood materially reduced, especially since some greenhouse rose varieties will produce shoots of which as many as 50 per cent will have aborted flower buds.
The hybrid tea greenhouse rose Better Times was used in this anatomical study of the development of both blind and floi;ering shoots. REVIEW OF LITERATURE
Blind wood has long been recognized by many investigators
(11, 14, and 19) as an economic loss to the rose grower. One of the first reports on the formation of blind wood was presented in 1859 by an anonymous writer (3) working with the variety Isabella Gray who stated, "It produced thirty strong shoots each of which terminated with blind ends". Since that time, some of the theories which have been advanced to explain the causes of blind wood are as follows: 1) an inherited characteristic (7), 2) a growth expression dependent upon the vigor or the stock (11), 3) the result of a hormonal condition
(13), 4) variations in light intensity and temperature (6), 5) light intensity and nutrition (23), factors other than light intensity and nutrition (8).
Heredity:
The first important work in blind shoots was done by Corbett (7).
One of the questions he was trying to answer was, "Do cuttings tend to perpetuate the individual peculiarities of the parent branch from which they are taken?" After 5 years of research he concluded in
1902, "that the tendencies manifested in a branch are perpetuated from generation to generation in plants propagated by asexual processes".
The basis for his theory was that rose plants propagated from flower ing shoots averaged 29.4 flowers per plant, while rose plants propaga- gated from blind shoots averaged 11.5 flowers per plant.
- 3 - - 4 -
Hubbell (12) was unable to substantiate the work of Corbett.
He budded axillary buds from mature flowering shoots onto flowering and blind shoots and axillary buds taken from blind shoots onto flowering shoots. He found that buds from flowering and blind shoots budded on flowering wood produced high percentages of flower ing shoots. Whereas, axillary buds taken from flowering shoots and budded on blind shoots produced a high percentage of blind shoots.
Hubbell concluded that the tendency for rose plants to produce blind- wood is not inherited but is a growth expression dependent upon the vigor of the stock. Therefore, according to Hubbell, the budding experiments indicated that the limitations of flower formation are based on the activity of the stock and not of the bud.
Activity versus Carbohydrabe-Nitragen Relationship:
In a pruning experiment by Hubbell (11) 97 per cent of the flowering shoots pruned back to the first, second, or third axillary bud from the tip remained flowering, while 97 per cent of the blind shoots pruned back in the same manner again became blind. He found that chemical analyses of blind shoots showed much greater percentages of non-colloidal nitrogen and insoluble carbohydrates than flowering wood; whereas flowering wood contained higher percentages of reducing sugars. In the spring of the year it became evident that there was an increase in the carbohydrate-nitrogen ratio. Since many blind shoots produced flowers in the spring, this caused a decrease in - 5 - blind shoots, and it was assumed that the increase in carbohydrate- nitrogen ratio brought about a condition xtfhich was favorable to
flower formation, Hubbell (11) stated that the budding experiments
combined with these pruning experiments indicated that blindness was not due to the impotency of the bud but was a result of the
stock,
In a review of Hubbell's (11) work on chemical analysis of
blind versus flowering wood, Kamp (13) expressed the opinion that
Hubbell had compared mature blind wood with immature flowering wood.
Also it was pointed out that Hubbell had analyzed the entire shoot
as a unit rather "than allowing for the possibility that gradients in
composition existed within the length of the stem. Kamp (13) there
fore, cut blind and flowering rose shoots into segments of varying
lengths and compared them. In the chemical analysis he measured free
reducing substances, total sugars, total nitrogen, and protein and non
protein nitrogen of comparable stem length segments. He concluded
that considering all these contradictory evidences, it was not
possible to see any clearly defined relationship between the number
of blind shoots and the chemical composition at least with respect to
the fractions analyzed in his study. He also stated that blindness
seems to be caused by a hormonal mechanism rather than by a nitrogen-
carbohydrate relationship. - 6 -
Karap (13) found upon examination of unshaded plants that regardless of variety, flowering shoots bore on the average more than twice the number of leaves as did blind shoots. Therefore, if blindness was attri buted to an insufficient number of leaves on a shoot due to a carbohydrate deficiency, systematic leaf removal should also cause blind shoots since it removes the photosynthesizing area. Shading should cause the same effect, reasoned Kamp, since it reduces the photosynthetic efficiency of the leaf surface. However, if blindness was due to an insufficient hormonal supply rather than a carbohydrate deficiency, it would be expected that defoliation would be more effective than shading in increasing blind shoots. Kamp's (13) results were as follows: 1) defoliation increased the total number of shoots, the number of blind shoots, and the percentage of blind shoots produced by the rose plant, and 2) shading had no effect on the total number of shoots or the number of blind shoots produced by the rose plant. Kamp's (13) conclusions on defoliation have also been reported by Daum (8).
After studying the correlation between the effects of light intensity and temperature with the occurrence of blind wood, Colacello (6) wrote that under conditions of optimum light intensity, temperature, nutrition, and other factors favorable for efficient photosynthesis auxin production would not be a limiting factor since the basic precursors of auxin are sugars which in turn are products of photosynthesis. Nutrition:
Hubbell (11) presented data showing that an increase in the soil nitrate supply decreased blind shoot production while a decrease in the soil nitrate supply increased blind shoot production.
Hasek (10) mentioning that the source of nitrogen had been a con troversial point for many years in the growth of blind shoots found by the use of solution culture that the source of nitrogen did not make a difference in the percentage of blind wood produced, provided proper con ditions were maintained. The nitrogen level appeared to have a more important role than the source of nitrogen.
Milne (16) presented data to show that when roses grew in soil at
80 parts per million of potassium, the total blind wood production was considerably lower than at either the 20 or 40 parts per million.
Working with solution cultures, Zink (23) found that as the nitrogen concentration in the solution was increased with corresponding increases in phosphorus and potassium, the percentage of flowering shoots increased.
Later he found that the level of phosphorus had little effect on the per cent of flowering or blind shoot production, but did give a slight in crease in flowers produced per plant. Contrary to the findings of Milne
(16), Zink found that an increase in potassium alone resulted in a corresponding increase in the percentages of blind shoots produced, while
the flowers per plant decreased as the potassium concentration increased. - 8 -
Different levels of concentration of three micrometabolic elements affected the percentages of blind shoots according to Zink (23). Both boron and manganese at low levels reduced the percentage of blind shoots in comparison to high amounts of these two elements. However, the re verse was true of magnesium where the high levels of this element decreased the percentage of blind shoots.
Daum (8) studied the relationship of nitrogen and potassium nutrition to the incidence of blind and flowering shoots as correlated with light intensity and temperature. The nitrate and potassium levels in the soil were adjusted so that high, medium, and low levels of each of the two elements were in combination with each other; thus nine combinations were used. He found that no significant results were obtained which might indicate that variations in the per cent of blind shoots produced were affected by the nutrient treatments. Therefore he concluded that factors other than those studied were responsible for the production of blind wood on the variety Better Times roses.
Light Intensity:
In 1929, an anonymous writer (2) stated, "With shorter days and gradually diminishing sunlight, the plants get too little light, and this alone causes roses to produce blind wood." The above statement reflects the opinion of many growers and has led to several investiga- of this problem. - 9 -
Hubbell (11) presented data showing that a decrease in monthly
illumination did decrease flower production but did not increase the
percentages of blind wood.
Bobula (4) reported using artificial illumination from 7:00 A.M. to
8:00 A.M. and from 6:00 P.M. to 10:00 P.M. and on cloudy days when the
light intensity directly above the plants was less than 2000 foot candles.
His data indicated that there was an increase in production of flowering
shoots from plants exposed to artificial illumination. This increase was not due to a reduced percentage of blind shoots but to the production of
a greater total number of flowering shoots. Some further observations
from the use of additional artificial illumination on blind shoot produc
tion were 1) that the use of artificial illumination was not effective in
stimulating the growth of terminal buds on pinched blind shoots, and 2)
that artificial illumination did not increase the productiveness of
shoots developed from the buds of pruned blind shoots...but did affect a slight increase in the productiveness of shoots developed from pruned flowering shoots.
From tests started in September 1945 and 1946, Kamp (13) reported
that there was an increasing number of blind shoots from September to
May; the high point being reached in May rather than in mid-winter as had previously been written in the literature. In the second year Kamp - 10 - recorded that increases in blindness began early in the fall when light conditions were still good. This trend lasted into the latter part of the spring well after the time when sunny weather prevailed.
Colacello (6) correlated light intensity and temperature and he reported low percentages of blind wood from late May to July during periods of high light intensity and temperature. Fluctuations in the total number of shoots produced and percentages of blind shoots were preceded by marked fluctuations in light intensity and temperature. He concluded that light intensity and temperature affected the productivity of blind and flowering shoots.
Zink (23) reported that a decrease in light intensity increased blind shoot production and decreased flowering shoot production. Zink stated that the production of blind shoots generally followed light intensity inversely and that blind shoot production on the temperature curve followed the reduced light curve very closely. However he con cluded that since plants in some plots had just as many blind shoots during high light intensities as during poor light intensities that not only light but nutrition was a determining factor in blind shoot pro duction and temperature played a secondary role.
Daum (8) found that variations in light intensity were not responsible for the production of blind wood on the variety Better Times roses. - 11 -
Anatomical:
The only extensive anatomical study of blind versus flowering shoots of roses was done by Hubbell (12). He stated that the growth of the blind shoot ceased after about 30 days. However the flowering shoot reached maturity in approximately 45 days.
However, Zink (23) compiled data on time between the start of bud development and maturity into two-month periods. He found that during
June and July the average total days to maturity including start of bud growth was 31.0 days. For the other three two-month periods that were studied he found; 1) August and September, 36.1 days, 2) October and
November, 43.5 days, and 3) December and January, 55.5 days. These findings were very similar to those of Laurie and Kiplinger (14) and
Post (19).
Hubbell (12) reported that floral differentiation in actively growing buds did not begin until 8 to 10 days after active growth had started, and complete formation of flower primordia was found at the end of the twenty-fifth day. The morphological differences in development between blind wood and flowering wood were stated by Hubbell as follows: "1) bland shoots were formed when the floral axis failed to develop a complete set of floral organs, 2) flower bud differentiation started about two days later in the blind shoots than in flowering shoots, 3) sepals and petals formed in the blind shoots, but no stamens or pistil primordia appeared, - 12 -
4) at the end of 30 days the undeveloped flowers showed signs of abortion and 5 days later it had completely aborted." In a few cases there were signs of abortion at the end of 28 days.
Bobula (4) presented data on the development of buds of flowering rose shoots of the variety Better Times. After collecting flowering buds at intervals of 14, 15, 16, 17, and 20 days after a cutback he found that
flower bud differentiation had occurred in buds which had grown actively for 14 to 18 days after they had been made to assume a terminal position.
Colacello (6) worked on the morphological aspect of blind wood, but he indicated his notes were merely preliminary in nature. He reported that a shoot was occasionally noticed that may have been in an intermedi ate stage between blind and a flowering stem. Such a shoot would grow to an average length of 6 inches and a flower bud would become visible, but instead of developing to maturity, the bud would become light green and abscise. He presented two photomicrographs of the same bud in which he showed that there was a marked development of sepal and petal primordia and the possible initiation of stamens and pistil primordia. However, below the bud an abscission layer had begun to form. MATERIALS AND METHODS
Plants:
Rose plants of the variety Better Times used in this study grew in the greenhouses of the Department of Horticulture and Forestry at the Ohio State University. A total of 364 five-year old started-eye plants were used in this study.
General Culture:
These plants grew in a standard type Lord and Burnham greenhouse,
100 feet long and 30 feet wide. A ground bed 93 feet long and 4 feet wide with a concrete, V-bottom separated from the soil level by a concrete layer, was used for the test. Half-tile was laid in the
V-bottom of the bench, running through its entire length, and gravel placed flush over the top of the tile to improve drainage and aeration.
Miami silt loam soil was then placed over the layer of gravel to within an inch from the top of the bench.
Maintenance of Soil Fertilizer Levels:
In this study roses were cultured according to the practices suggested by Laurie and Kiplinger (14). The fertilizer program carried out up to the time the plants were cutback was evaluated every two weeks by the Spurway tests (20) for nitrates, phosphorus, potassium, and calcium. The pH was determined by means of a Beckman pH meter, Model H,
- 13 - - 14 - and soluble salts were ascertained every four weeks through the use of the solu-bridge. (Industrial Instruments Company Model R.D. 15).
Water was withheld for one watering; then the plants were cutback
March 7, 1952. No fertilizer was added following the cutback until the plants resumed normal growth.
Sampling:
When this problem was proposed, it was the intent of the author to take one set of shoot apices that would ultimately have been flowering shoots and another set of shoot apices that would become blind shoots.
Then after proper sectioning and staining the anatomical development of these two types of shoot apices would be investigated. However, for a great part of this development it was impossible to be absolutely certain whether the shoot apices being collected ware from blind or flowering shoots. Chan (5) stated that it was fairly certain that those shoots which turn green after 25 days would be blind while the more vigorous flowering shoots would still have a dark reddish tinge. Although this did give an approximate idea which shoots would be blind or flowering after 25 days, it did not aid in the selection of blind apices from the first to the twenty-fifth day.
It is a common practice in the spring of the year to cutback rose plants when forcing them under glass. One reason for this is to reduce the height of the cutting zone so that the florist may more easily cut - 15 - the flowering shoots. When the rose plant is cutback to a height of 18 to 30 inches depending upon the age of the plant, it has been noted that many axillary buds will break. In most cases the uppermost axillary bud on each shoot will develop into a heavy-caned flowering shoot which become the upper framework of the plant for that year. However, many of the sub-axillary buds which also break along with the uppermost axillary buds become blind. After many of these sub-axillary buds at the upper end of the shoot grow and become blind, the appearance of this blind wood together gives rise to the term "birds nest". It was decided that the rose plants in this study would be cutback, and it was anticipated that the apparently dormant terminal axillary buds would assume the terminal position and produce a large percentage of flowering shoots while apparently dormant sub-axillary buds would develop into a large percentage of blind shoots. This was studied by Chan (5) who tabulated the figures on the percentage of flowering versus blind shoots of five-year old plants which had been cutback. His study showed that of the total number of shoots produced, blind shoots occurred in the approximate percentage of
66 percent. This figure was based on a total of 4,500 shoots. Therefore, it was reasonable to assume that if the terminal buds had not been counted in this study, the percentage of blind shoots would have been still higher. Under these conditions it can be assumed that at least 2 out of every 3 sub-axillary buds sampled would have developed into blind shoots. - 16 -
Sampling began the first day (March 8, 1952) after the plants were cutback. Ten terminal and ten axillary buds were sampled each day for
35 days from the cutback plants.
Embedding and Cutting Techniques:
The bud samples used for microscopic studies consisted of stem tips measuring three-eighths inch or less in length. When necessary, all possible leaves were removed without damaging the shoot apices. Care was taken in selecting stem tips so that only representative samples were used. Each sample consisting of ten shoot tips of both the uppermost and sub-axillary buds was placed immediately into individual one-ounce wide- mouth French square bottles containing FPA (5 millileters of propionic acid, 5 millileters of formalin, and 90 millileters of 50 per cent ethyl alcohol) fixing-killing solution. In order to remove air trapped in the stem tips and to enhance the penetration of the killing solution into the stem tissues, the plant material was placed in a vacuum apparatus similar to that described by Wittlake (21) for approximately 24 hours during the killing process. A partial vacuum of about 10 millimeters of mercury was used. Shoot apices were run through an ethyl alcohol and a xylene series and finally embedded in a summer grade, rubber-paraffin embedding mass.
Longitudinal, serial sections of the shoot tips were cut at 8 microns on a rotary microtome. - 17 -
Staining:
The staining procedure used was an adaptation of that described by Popham, Johnson, and Ghan (18) and included the following steps:
1. Dip in xylene andleave 10 minutes.
2. Bring slides to 50 per cent ethyl alcohol.
3. Stain in 1 per cent safranin for 3 minutes.
4. Dip in tap water.
5. Dip in each of the following: 50%, 70%, 85%, 95%, and absolute ethyl alcohol.
6. Dip in saturated solution of analin blue in methyl cellosolve and leave for 20 minutes.
7. Dip in absolute ethyl alcohol to remove excess analin blue from the sections.
8. Dip in rinse solution.
9. Dip in clearing solution andleave 10 minutes.
10. Dip in mixture of 90 per cent xylene and 10 per cent absolute alcohol.
11. Dip in two successive stender dishes of xylene and leave for 10 minutes in each dish.
12. Mount in piccolyte.
13. Cure slides in 60° C. oven for two days.
The analin blue stain suggested by Popham et. al., (18) was not satisfactory for this material. It was found that a stock solution could be made by adding three grams of analin blue to 125 cc. of absolute alcohol and 125 cc. of methyl cellosolve. From this stock solution a - 18 - satisfactory staining solution was made by adding 50 per cent by volume of the analin blue stock solution to an equal volume of clove oil.
Since Hubbell (12) stained his serial sections in safranin-light green, the author used the same staining procedure in order to compare his results with that of the author. RESULTS
A sequence of the initiation and differentiation of the flowering
and blind rose apices were obtained from the serial sections of the
sampled buds. For clarity these are presented in three groups:
1) vegetative, 2) flowering, and 3) blind.
Vegetative Development
When either an ultimately flowering or blind shoot first develops,
the apex is in a vegetative condition. Fig. 1 is a photomicrograph of
a terminal bud which presumably would have been a flowering shoot
(terminal), while Fig. 2 is a photomicrograph of a shoot in a vegetative
condition from a stem that presumably would have been blind (axillary).
- 19 - - 20 -
Fig. i. A vegetative stem apex from a shoot that presumably would have been flowering (terminal). The general outline of the apical meristem was always conical when the shoot was vegetative. A vegetative stem apex from a shoot that - presumably would have been blind (axillary). Note that the appearance of the apical meristem is identical with that of a flowering shoot (Fig. 1). - 22 -
Initiation and Subsequent Development of the Flowering Shoot Apex
The development of the apical meristem of the flowering shoot is shown in figures 3 through 9 which follow. - 23 -
Fig, 3. The first evidence of reproduction in a presumably flowering shoot is shown by the development of enlarged areas at each side of the meristem and a flattening at the mid-point of the meristem. This occurred approximately 7 days after cutting back. - 24 -
Fig. 4. An early stage of sepal primordia indicated by the humps at the flanks of the apical meristem. There are 5 sepals in the rose and this number of humps were found in serial sections. Sepal primordia were first observed 11 days after the cutback. - 25 -
Fig. 5. A later stage of development of sepal primordia. An axillary bud is shown in the lower right hand corner and the beginnings of the procambial strands may be identified by the acropetally dividing cells just to the left of the bud. - 26 -
Fig. 6. An early stage of petal primordia which appeared 13 days after the cutback. These show as the rounded protuberances at either flank of the apical meristem. The sepals enclosed the meristematic tip in this view and procambial strands in the sepals are clearly discernible. - 27 -
[n Jmmwi ' m i I ' MA
Fig. 7. A later stage of petal primordia formation occurring about 15 days after the cutback. Two of these primordia flank the apical meristem and others may be observed immediately above the apical meristem. The sepals in the photomicrograph no longer enclose the meristem and are at right angles to its axis. - 28 -
r a p & w ' § «
Fig.8. An early stage of pistil primordia which appear as small irregular humps at the location formerly occupied by the apical meristem. This stage occurred approximately 22 days after cutting back. Flanking these humps is the primordial stage of the stamens. - 29 -
A:
Fig. 9. A later stage of pistil primordia development. Stamens are distinguishable by rudimentary anthers and filaments. Two of them are clearly visible at the right and left of the center of the photomicrograph. - 30 -
Initiation and Subsequent Development of the Blind Shoot Apex
The development of the apical meristem of the blind shoot is shown in figures 10 through 20 which follow. - 31 -
i » « i i
Fig. 10. The first evidence of reproduction in a presumably blind shoot shown by the develop ment of a flattened apical meristem. - 32 -
Fig. 11. The formation of sepal primordia has occurred on the flanks of the apical meristem of this presumably blind shoot. - 33 -
Fig, 12. The first evidence of disintegration of tissue in a presumably blind shoot can be seen in the darkly stained areas of the left sepal. Both epidermal layers as well as the second and third rows of cells adjacent to each epidermal layer have begun to disintegrate. The same process appears to be occurring in the right sepal, but at a slower rate. Petal primordia are seen flanking the apical meristem. No disintegration was noted in any section prior to the formation of the petal primordia. This is con sidered the first stage of abortion. - 34 -
Fig. 13. Further disintegration of the sepals as shown by vacuolation of the cells in the procambial strands and the formation of a necrotic band in each sepal in approximately the third layer of cells below the epidermis. This band extends from the tip of the sepals down the flanks into the area below the apical meristem. What looks like another band or similar area is beginning to form below the apical meristem and proceeds across the bud at right angles to the epidermal cells of the sepals. This is the intermediate step in the abortion of the rose apex. - 35 -
Fig. 14. Complete abortion of the rose apex before stamen primordia were visible. Note the complete disintegration of tissue below the darkly stained cells. - 36 -
Fig. 15. Stamen primordia are shown on the flanks of the apical meristem. Even at this rather fully differentiated stage of the flower bud, necrotic bands are clearly evident in the sepals and petals which is indicative that the shoot will be blind. - 37 -
Fig. 16. Disintegration of cells well below the apical meristem is clearly evident at the stamen primordial stage. - 38 -
Fig. 17. At an early stage of pistil formation, necrotic bands are clearly evident in the sepals and petals. - 39 -
Fig. 18. Disintegration of the cells below the apical meristem has occurred at a very early stage in the differentiation of pistil primordia. Fig. 19. An aborting rose apex showing necrotic bands in the sepals and petals and cell disintegration. - 41 -
Fig. 20. An enlarged photomicrograph of the abscission or disintegration zone of the bud shoiirci in fig. 19. - 42 -
Relative Speed of Development of Stem Apices:
The time required for the apices to reach a given stage of development, is shown in table 1.
Table 1. The number of stem apices at any given stage of development at the various times of sampling in relation to the position ______of the bud through the petal initiation s t a g e . ______Flat tened Sepal Petal Sampling Vegetative apex initiation initiation day Ter Axill Ter Axill Ter- ; Axill Ter Axill minal ary minal ary minal ary minal ary
2 2 3 3 2 4 3 3
5 3 4 6 6 9 7 4 8 1
8 4 9 3 9 1 8 4 1 10 8 5
11 5 2 3 3 12 4 4 1 13 1 1 7 3 14 1 1 8 5 15 4 6 2 16 1 3 4 5 17 4 6 18 3 9 DISCUSSION
When a rose shoot is cutback, the uppermost axillary bud becomes the terminal bud and this shoot will usually produce a flower. However, most of the shoots developing from the accompanying axillary buds will be blind. Due to the apical dominance of the terminal bud, it will develop sooner, and therefore the anatomical changes, from the vegetative to the reproductive phase will occur before these changes take place in the lower axillary buds. But, once the axillary bud has started to grow, the differentiation of this axillary apex that will become blind proceeds at the same rate from one primordial phase to another as the terminal flowering apex until physiological conditions are established which cause the apex to become blind, (Table 1). This difference in development of the shoot depending upon the position on the stem probably accounts for
Hubbell's (12) statement that flower bud differentiation was two days later in the blind shoot than in the flowering shoot which he referred to as "sluggish differentiation". His term gives the concept that every stage in the anatomical development of the blind shoot is retarded, or even more, that blindness is predetermined from the start of growth.
However, there is evidence that the development of the blind apex was similar anatomically to that of the flowering apex (in relation to time for the initiation of succeeding anatomical phases) until some condition,
- 43 - 44 -
(internal or external), causes the eventual abortion of the bud, figures
1, 2, 3, 5, 10, 11 and Table 1. The flowering and blind shoot will there fore develop similarly up to the time when cells in the sepals or sepals and petals begin to disintegrate.
Hubbell apparently did not take into consideration the position of the axillary bud in relation to the terminal bud when using the term sluggish diferentiation. Since the degree of apical dominance determines the bud development sequence on a shoot, it is logical to assume that the delay in axillary bud groxAih is due to the position of the bud on the shoot. This is especially so since there was no observable difference betx^een the anatomy of the flowering apex and of those axillary buds which had not yet shown any observed symptoms of blindness.
In the author's opinion, no axillary buds gave any indication of becoming blind until the formation of petal primordia, Fig. 12. There fore, it was assumed that up to and including the formation of sepal primordia, there was no essential difference in the developmental anatomy of the blind or flowering shoot apex.
Evidence of Abortion
Hubbell (13) reported that in a blind apex, sepal and petal formation vias accompanied by broadening of the receptical cup. At this time, there was no evidence of pistil or stamen formation, and according to Hubbell the bud shoxved clear signs of abortion. However, no indication \
Hubbell (12) used the safranin-light green method of staining and did not report any disintegration of cells or appearance of necrotic bands in sepals or petals. In this study much of the staining was the safranin- analin blue. To determine whether the staining material used might have caused differences in interpretation, a series of slides in this study were stained using Hubbell's method. Cell disintegration and necrotic bands were noted with safranin-light green just as clearly as with the safranin-analin blue dyes indicating that Hubbell must have overlooked this early stage of abortion.
Time for Abortion to Take Place
In this study there was a minimum of importance given to the number of days for any anatomical change to occur, but rather the significance of differences were stressed between the anatomical changes in the flowering apex. Therefore the anatomical changes appear to be the only true criterion of blindness since four to eight weeks are necessary in order for a rose shoot to develop from the start of growth to maturity, - 46 -
depending upon the season. Therefore, anatomical changes would be
affected by any seasonal change in growth. This was clearly shown
by Zink (23) who observed variations in growth at different seasons.
Differences in time of abortion between this study and that of a Hubbell (12) are considerable. In this study abortion is associated
with disintegrating cells which were first noticed 19 days after growth
started. However in Hubbell's study abortion was associated with the
development of an abscission layer which was reported 34 days after
growth started.
Initiation and Subsequent Development of the Blind Shoot Apex
For purposes of convenience it appears possible to classify abortion
into 3 development stages. These are 1) primary stage or cell disintegra
tion, 2) intermediate stage or band necrosis, and 3) mature stage or
formation of an abscission layer. Not all of these stages are shown
in the photomicrographs.
The first signs of abortion occurred when the bud was just beginning
to develop petal primordia (Fig. 12). The disintegration of tissue could
be seen in both epidermal layers of the sepals, and this disintegration
continued down both sepals into the bud below the apical meristem. An
interesting point here is that the last tissue to become disorganized
was that of the apical meristem or that tissue which had just differenti ated from the apical meristem into the pistil primordia as shown in Fig. 18. - 47 -
The early disintegration of tissue was followed by necrotic bands of the intermediate stage, figures 13, 15, 16, 17, and 18. A point to be considered here was that there was never an abscission layer which was not first preceded by these necrotic bands.
Following this necrotic band stage was the actual formation of the abscission layer and abortion of the apex, (figures 14 and 16).
The primary, intermediate, and mature stages of blind wood could be found in the petal primordia stage and most of these stages were also found in buds which had developed to the stamen and pistil primordia stage, (figures 15, 16, 17, and 18). When buds had progressed to the stamen or pistil primordia stage, cells of the petals as well as the sepals gave the first evidence of cell disintegration. This is therefore in complete disagreement with the findings of Hubbell who could not find any blind apices in buds which had progressed to the stamen or pistil primordia stage. The evidence in this study indicates that the rose apex of the variety Better Times can become blind at any time from petal through pistil formation. No blind apices were found before the petal primordia stage.
Blindness
The foregoing discussion concerns the question of the causes of blind wood. The author is inclined to agree with Kamp (13) that some hormonal mechanism is involved in blind wood formation because of the presumed absence of some essential substance for continued growth. That nutrition in itself is apparently not the cause of blind wood has been shown by
Daum (8). In addition, work by Lindstrom and ICiplinger (15) showed that fertilizer variations had no significant effect on blind wood production
In some unpublished data the author found that by ringing terminal shoots during the winter before the buds began to develop or before the immature shoot is more than 3 or 4 inches long, that 100% of these shoot will become blind. Kamp (13) found that during the period of shoot development there was a movement or translocation of free reducing substance and total sugars into the. new shoots and thi') moved gradually toward the tip of the stem as the flower developed. It is therefore possible that a sugar concentration in relation to a hormone or hormone percursor is necessary before the concentration of that particular hormone in question is present in quantities which can complete the entire process of floral reproduction. SUMMARY AND CONCLUSIONS
Serial sections of blind and flowering shoots of the rose variety,
Better Times, were examined microscopically to discover the stages at which signs of abortion could be distinguished.
1. The transition from the vegetative to the reproductive stage
was considered -to be the flattening of the apical meristem,
2. The first signs of abortion of the apex was disintegration
of cells in the sepals or sepals and petals.
3. The next stage in the abortion of the bud v?as characterized
by the appearance of necrotic bands in the sepals or sepals
and petals.
4. The final stage was the appearance of an abscission layer
in the stem below the pistil primordia.
5. Blind apices were found from the time of petal primordia up
to and including pistil formation.
- 49 - BIBLIOGRAPHY
1. Agriculture Census 1950. Vol. V. Special Reports. Part 1. Horticultural Specialties.
2. Anonymous. Blindness in roses and pompons. Flor. Rev. 65: No. 1674. 28. Dec. 26, 1929.
3. Anonymous. Roses. Gard. Cliron. and Gaz.* 1859: 28. June 18, 1859.
4. Bobula, P.F. 1939. Some preliminary observations on blindness of forced roses. Unpublished Master's Thesis. Ohio State University.
5. Chan, A,P. Personal communication. 1952.
6. Colacello, F.J. 1940. Correlation studies of light intensity and temperature with the occurrence of blind wood of Better Times Roses. Unpublished Master's Thesis. Ohio State University.
7. Corbett, L.C. 1902. Improvement of roses by bud selection. Mem. Hort. Soc. New York. 1: 93-101.
8. Daum, P.L. 1951. Studies of the relationship of nitrogen and potassium nutrition to the incidence of blind and flowering shoots and the total linear growth as correlated with light intensity and temperature. Studies on the effect of various pinching and cutting methods on the incidence of blind and flowering shoots aid total linear growth as correlated with light intensity and temperature. Unpublished Master's Thesis. Ohio State University.
9. Grove, L.C. 1933. Blind wood in roses as influenced by certain practices. Unpublidied Master's Thesis. Iowa State College.
10. Hasek, R.F. 1940. Nutritional studies with the rose. Unpublished Master's Thesis. Ohio State University.
11. Hubbell, D.S. 1934. Causes of blindwood in roses. Plant Physiol. 9: 261-282.
12. , D.S. 1934. A morphological study of blind and flowering rose shoots with special reference to flower-bud differentiation. Jour. Agr. Res. 48: 91-95.
- 50 - - 51 -
13. Kamp, J.R. 1947. The incidence of blindness in Better Times roses. Unpublished Doctor of Philosophy Thesis. Ohio State University.
14. Laurie, Alex and D.G. Kiplinger. 1948. Commercial Flower Forcing. The Blakiston Co., Philadelphia, pp. 550.
15. Lindstrom, R.S. and D. C. Kiplinger. 1956. Blind wood of Better Times roses as affected by selection of stock and nitrogen and potassium nutriton. Proc. Amer. Soc. Hort. Sci. 66.
16. Milne, C.G. 1940. Nutritional studies with the rose. Unpublished Master's Thesis. Ohio State University.
17. Meyer, B.S. and D.B. Anderson. 1952. Plant Physiology. Van Nostrand Co., New York, N.Y. pp. 784.
18. Popham, R.A. , T.S, Johnson and A.P. Chan. 1948. Safranin and aniline blue with delafields hematoxylin for staining cell walls in shoot apexes. Stain Tech. 23: 185-190.
19. Post, Kenneth. 1949. Florist Crop Production aid Marketing. Orange Judd Publ. Co. Inc., New York, N.Y. pp. 891.
20. Spurway, C.H. 1949. Soil testing, a practical system of soil diagnosis. Mich. Agr. Exp. Sta. Tech. Bull. 132: 1-39.
21. Wildon, C.E. 1946. Garden Roses. Mich. Agr. Exp. Sta. Bull. 222: 22-62.
22. Wittlakej’ E.B. 1942. An efficient vacuum apparatus for micro- technic. Ohio Jour, of Sco., 42: 65-69.
23. Zink, E. 1950. Studies of the blind wood of roses as affected by nutrition. Unpublished Master's Thesis. Ohio State University. AUTOBIOGRAPHY
I, Richard Stadden Lindstrom, was born in Cleveland, Ohio, March
5, 1927. I received my secondary school education in the puolic schools of Columbia Station, Ohio. My undergraduate training was obtained at
the Ohio State University, from which I received the degree Bachelor of
Science in Agriculture in 1950 with a major in Horticulture.
I enrolled in the graduate school of the Ohio State University where I received the degree Master of Science in 1951 and continued with graduate work until 1953. During the time I was in graduate school I held a Fellowship which was granted by Roses, Incorporated. In September
1953, I was appointed Instructor of Horticulture at Michigan State Univer sity, where I now hold an appointment for half-time teaching and half- time research. The remainder of my work for the degree of Doctor of
Philosophy was done at Michigan State University, where I worked under an off-campus research agreement with the Ohio State University.