Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1-1-1984 The oler of water stress and ethylene in Ficus benjamina L. leaf abscission William Richard Graves Iowa State University
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Recommended Citation Graves, William Richard, "The or le of water stress and ethylene in Ficus benjamina L. leaf abscission" (1984). Retrospective Theses and Dissertations. 17442. https://lib.dr.iastate.edu/rtd/17442
This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The role of water stress and ethylene in Ficus benjamina L. leaf abscission
by
William Richard Graves
A Thesis Submitted to the
Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE
Major: Horticulture
Signatures have been redacted for privacy
Iowa State University Ames, Iowa
1984 ii
TABLE OF CONTENTS
Page
INTRODUCTION
REVIEW OF LITERATURE 3
SECTION I. WATER STRESS, ETHYLENE, AND FICUS 9 BENJAMINA L. LEAF ABSCISSION
INTRODUCTION 10
MATERIALS AND METHODS 12
RESULTS AND DISCUSSION 14
SECTION II. EFFECT OF WATER STRESS ON FICUS BENJAMINA L. 19 LEAF ABSCISSION
INTRODUCTION 20
MATERIALS AND METHODS 21
RESULTS AND DISCUSSION 23
SECTION III. EFFECT OF WATER STRESS ON 32 ENDOGENOUS ETHYLENE CONCENTRATIONS IN FICUS BENJAMINA L. LEAVES
INTRODUCTION 33
MATERIALS AND METHODS 35
RESULTS AND DISCUSSION 37 iii
SUMMARY AND CONCLUSIONS 45
LITERATURE CITED 47
ACKNOWLEDGEMENTS 52
APPENDIX A. INITIAL WATER STRESS AND ETHYLENE EXPERIMENTATION 53 WITH FICUS BENJAMINA L.
APPENDIX B. GAS CHROMATOGRAPH CONDITIONS AND CDS-111 PROGRAM 55 INTRODUCTION
The foliage plant industry in the United States has experienced explosive growth during the past two decades. As tropical plants have become increasingly common in offices, homes, and shopping centers, consumers have become more aware of problems associated with their care. One of the most popular indoor plants in northern regions of the
United States is Ficus benjamina L., the Weeping Fig. Its dark green, delicate foliage and graceful tree-like habit have made it a favorite among interiorscapers. Along with realizing its aesthetic value, many consumers have encountered problems associated with its indoor culture. Perhaps the most common complaint is that of leaf abscission, especially when rapid environmental changes occur.
The majority of commercially available Ficus benjamina plants in northern regions of this country are grown in the South and shipped north. Investigations of the leaf abscission problem have focussed on production and shipping practices which contribute to defoliation.
This research has resulted in light acclimatization, a practice involving a reduction in the irradiance during the final weeks of the production schedule. Extensive use of this technique by commercial growers partially has alleviated the defoliation problem.
Although light acclimatization practices have alleviated extensive Ficus benjamina leaf abscission, the problem has not been eliminated. This research was designed to examine the relationship 2 between leaf abscission induced by water stress and the internal concentration of ethylene (C H ), a plant growth regulator often 2 4 associated with the abscission of plant parts. The author feels concentrations of endogenous H associated with the abscission c2 4 process are similar, regardless of whether abscission is induced by water stress or by differentials in the irradiance. Thus, it is hoped that the data presented will be of value to future researchers who may use any one of several procedures to induce leaf abscission.
The objectives of this project were: 1) to determine the amount of leaf abscission resulting from different degrees of osmotically induced water stress, and 2) to associate the process of leaf abscission with internal concentrations of C H • 2 4 3
REVIEW OF LITERATURE
Water Stress and Leaf Abscission
Water is essential for plant growth, and its supply plays a crucial role in almost every plant function. A lack or overabundance of water supplied to plant root systems results in stress. Such water stress has been shown to result in numerous morphological and physiological adjustments (20). One such adjustment is premature leaf shedding, a logical response, as it serves to decrease transpiration during times of summer drought, in response to arid winds, and during the wet and dry seasons of the tropics (33).
Studies involving the effects of drought stress on abscission have shown bolls and leaves of cotton abscise in an increasingly linear manner as leaf water potentials decrease from -10 to -24 bars
(39). Leaf abscission after water stress of cotton generally involves older foliage first (39) and abscission occurs after water deficits are relieved by irrigation (30, 39). Work with Ficus benjamina has demonstrated water deficits induce leaf abscission (25, 28, 49, 50,
51, 52). Peterson, Sacalis, and Durkin showed that over 40 percent leaf abscission follows exposure of Ficus benjamina to osmotically induced water stress and that abscission of older foliage is most common (50, 51, 52). Further work has revealed that soil moisture stress during production of Ficus benjamina reduces the transpiration rate, shoot and root dry weights, leaf fresh weight, and leaf area, but when placed under interior conditions, plants which had been 4 irrigated more frequently during production experienced greater leaf abscission (25). Others have shown water stress of Ficus benjamina promotes reduced dry matter accumulation and reduced stem caliper
(28).
Studies involving plant water stress employ any one of several methods for stress induction. Frequency of irrigation and the introduction of various osmotica to roots of plants grown in an aqueous medium are common techniques. Solutions of high molecular weight forms of polyethylene glycol (PEG) frequently are used to evoke a constant degree of water stress which mimicks closely conditions expected in soil (31). Osmotic potentials of aqueous solutions of PEG
6000 have been related to PEG concentration (43). High molecular weight forms of PEG, generally those over 3000 daltons, do not penetrate plant tissues (42), an important advantage over other osmotica. Others have reported side effects associated with the use of PEG including reduced oxygen availability to the roots (41) and absorption into damaged roots (34). Therefore, although the use of
PEG to induce water stress can provide a degree of control difficult to attain by withholding irrigation, its use should involve caution and awareness of possible side effects.
Ethylene and Leaf Abscission
Ethylene (C 2H4) is a simple, unsaturated, hydrocarbon compound that exerts a major influence on virtually every aspect of plant growth and development. Nearly every plant tissue appears to be 5 capable of H production (60) which is thought to proceed via the c2 4 following conversions: methionine to S-adenosyl methionine (SAM), SAM to 1-aminocyclopropane-1-carboxylic acid (ACC), and ACC to c2H4 (3). Although the elucidation of its biosynthetic pathway has come only recently, the profound effects of H on plant growth and c2 4 development have been demonstrated beginning in 1901 when Neljubov described the now-classic "triple response" of etiolated pea seedlings. This response includes increased radial expansion of stems, inhibition of stem elongation, and horizontal orientation of
stems with respect to gravity (47).
H , after its discovery in illuminating gas, first was found to c2 4 affect leaf abscission by Doubt in 1917 (16). In 1952, Jackson found
that applied H was capable of inducing premature senescence in c2 4 cotton. The symptoms of this senescence included leaf abscission, and
led him to postulate that H played an active role in the abscission c2 4 process ( 21).
More recently, numerous studies have focussed on the relationship
between C H and leaf abscission (1, 6, 15, 23, 27, 44, 48). Jackson 2 4 and Osborne found large amounts of H production in tissue at a c2 4 distance from the abscission zones of leaves on 3 species during senescence (23). After other studies revealed cellulase (19) and
pectinase (46) activity increased in the abscission zones of senescent
leaves, Addicott postulated that H may stimulate production of c2 4 energy-rich compounds needed for the synthesis of these enzymes (4).
The relationships between H , other plant growth regulators, c2 4 6 and leaf abscission have been explored (4, 35, 36). Abeles and
Rubinstein (2) and Rubinstein and Abeles (58) concluded that
endogenous H affects the abscission process after showing H c2 4 c2 4 evolution is related to levels of auxin, and that partial removal of
accumulated H in bean explants slowed leaf abscission. In 1968, c2 4 Burg suggested that H promotes leaf abscission by causing reduced c2 4 auxin levels and by stimulation of cellulase activity in cells of the
abscission zone (11). Beyer found that applications of
naphthaleneacetic acid to leaves of H -treated plants prevented leaf c2 4 abscission The fact that H generally promotes abscission of a (6). c2 4 plant's older foliage first (45, 48) presumably is due, at least in
part, to lower levels of auxin present in leaves as they age (45).
The importance of the effect of H on leaf abscission in c2 4 commercial situations has been explored by Cunningham and Staby (15)
who showed that H concentrations of 1 pl/1 inside containers used c2 4 in simulated shipping experiments resulted in up to 100 percent leaf
abscission of ornamental lime plants. The significance of these
results is questionable because they failed to detect H in the c2 4 atmospheres of containers of abscission-plagued lime plants shipped
from Florida to Ohio. Work with Ficus benjamina has shown that
ethephon soil drenches caused morphological changes and increased leaf
abscission, especially when plants first had not been light
acclimatized (27). 1
Water Stress and Ethylene
Investigations showing both water stress and H affect leaf c2 4 abscission have provided incentive to study their relationship. In
1971, McMichael, Jordan, and Powell reported that internal levels of
H may mediate leaf abscission of water stressed cotton plants, and c2 4
they found that production of c2H4 in intact cotton petioles increased within hours after water deficits developed (39). Others have shown
that cotton seedlings subjected to water stress are more prone to
H -mediated leaf abscission than nonstressed plants (30). In this c2 4 study, treatment with 2.0 to 3.2 ~1 H /l caused the threshold plant c2 4 water potential capable of induction of abscission to rise from -17 to
-1 bars. Thus, the researchers concluded that water stress
predisposes tissue to C H action (30). 2 4 Studies involving similar phenomena in other plant systems
include a 1974 report which showed that waterlogging or drought stress
of Vicia faba resulted in increased endogenous c2H4 levels and that higher H concentrations were correlated with greater leaf and c2 4 flower abscission (17). In a more recent study, water stress of
asceptically cultured plum leaves caused increased H production, c2 4 and H concentrations were maximal at 50 percent water loss (32). c2 4 Increased endogenous levels and production rates of H also have c2 4 been observed in detached 'Valencia' orange leaves when stressed by
placement in an atmosphere containing 55 percent relative humidity
(5). Subsequent relief from 10 to 20 hours of such stress caused H c2 4 concentrations to return to levels observed in nonstressed leaves. In 8
1977, Wright observed that excised wheat leaves produced more H c2 4 when water stress was applied, and he found that the amount of H c2 4 produced was related to stress severity (59). Maximum H production c2 4 was associated with leaf water potentials of -12 bars. In addition, at all stress levels, c2H4 production was greatest between 135 and 270 minutes after stress was applied (59).
These findings created an interest in the mechanism controlling
the water stress-C H relationship. In 1976, Jackson and Campbell 2 4 reported waterlogging of tomato reduced dissolved oxygen levels in soil water and that waterlogging resulted in epinastic growth of
tomato petioles and increased H concentrations in the shoot (22). c2 4 In 1978, Jackson, Gales, and Campbell found a 4- to 6-fold increase in
shoot c2H4 production in waterlogged tomatoes, though waterlogging did not cause decreased leaf water potential (24). Since these reports,
Bradford and Yang have demonstrated that levels of 1-
aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of
H , increased in roots and shoots of waterlogged tomatoes. Under c2 4 such conditions, ACC from the roots is thought to be transported
through the xylem to the shoot, serving as a "signal" to the shoot
system and leading to increased H production (8, 9). Further c2 4
research involving treatment with c2H4 inhibitors during waterlogging of tomato (10) supports the Bradford and Yang hypothesis. Subsequent
research has shown that pretreatments of water-stressed, excised wheat leaves with benzyladenine or indole-3-acetic acid caused increased
H production and increased endogenous ACC levels (37). c2 4 9
SECTION I. WATER STRESS, ETHYLENE, AND FICUS BENJAMINA L. LEAF ABSCISSION 10
INTRODUCTION
One of the more popular plants used for interior landscaping is
Ficus benjamina L. Despite its widespread use, it has gained a reputation for being slow to adjust satisfactorily to sudden changes in environmental conditions (14, 25, 26, 50, 53, 54, 55). The most common problem associated with this adjustment has been the abscission of a large portion of its foliage after introduction to a new setting.
The leaf abscission itself generally is preceded by a color change of the affected foliage from deep green to a bright golden yellow.
Researchers have addressed the problem of Ficus benjamina leaf abscission. Their results have revealed that, if plants are grown under 20 or 60 percent sunlight during the final 5 or more weeks of the production schedule, significant reductions in leaf abscission result (14). Consequently, the practice of light acclimatization has been accepted widely by commercial foliage growers and, to some extent, has alleviated the problem.
Although light acclimatization practices have alleviated the problem of complete defoliation of Ficus benjamina, leaf abscission has not been eliminated. For this reason, further research has focussed on the role of water stress in defoliation (25, 49, 50, 51,
52). Researchers have found that withholding water caused leaf abscission with plants grown in sand as did additions of various amounts of polyethylene glycol (PEG) 6000 to the nutrient solutions in which plants were grown hydroponically (51). The findings of this 11 research indicate that both light and water stress affect the defoliation of Ficus benjamina. Although leaf abscission can be minimized by light acclimatization and maintenance of proper water
status, the question of what controls defoliation internally remains
unanswered.
This research was designed to examine the relationship between
leaf abscission of Ficus benjamina induced by four levels of water
stress and the internal concentration of H , a plant growth c2 4 regulator often associated with abscission of plant parts. Section I
of this thesis deals with experiments involving concentrations of PEG
8000 used in preliminary work only. 12
MATERIALS AND METHODS
Terminal cuttings 12 to 18 em long taken from plants which had originated from a common parent plant were used for all experiments.
After rooting in perlite for approximately two weeks, plants were shifted to growth chambers and grown hydroponically in 500 ml mason jars containing full-strength, aerated Hoagland's Solution (18). Each plant was supported individually by a 70 mm DiSPo~ Plug. Chamber conditions included a 14-hour daylength with an irradiance of approximately 325 ~E/sec/m 2 at 25° C. During dark hours, the temperature was reduced to 20 0 C. Relative humidity was approximately
65 percent. After five weeks of conditioning in the chamber, either
40 or 48 uniform plants, depending on the experiment, were selected and placed into four treatment groups of equal size. Plants generally had 40 to 100 leaves at the time of treatment.
To determine the effect of osmotically induced water stress on leaf abscission, plants were shifted into nutrient solution that contained either 0, 300, 350, or 400 g/1 of PEG 8000. Each treatment contained 10 plants chosen randomly. These PEG concentrations caused considerable leaf abscission during preliminary experimentation and resulted in calculated solution osmotic potentials of -3, -13, -18, and -24 bars, respectively, using the equation of Michel and Kaufmann
(43). Plants remained in this system for 48 hours and then were shifted back into nutrient solution containing no PEG. Leaf abscission was monitored daily for one week. 13
Analysis of variance was performed using SAS to test for statistically significant differences between the treatments. The
Least Significant Difference (LSD) at the 5 percent level for differences between mean values was applied when F-tests were significant.
Identical PEG exposure procedures were used in separate experiments to determine internal c2H4 concentrations associated with the leaf-drop phenomenon. Twelve plants were assigned randomly to each of the four PEG levels. Each PEG treatment level contained three replicates, and each replicate contained four plants. From the time
PEG exposure began, and, at 24-hour intervals thereafter, three leaf blades and their attached petioles from each plant in each replicate were removed and analyzed for internal H concentration. Each 12- c2 4 leaf sample consisted of one young, one intermediate, and one older leaf taken from three different regions of each plant in each replicate. The leaves from each replicate were grouped, weighed, and placed in an apparatus designed to extract the atmosphere from plant tissue (7). Gas samples were analyzed every 24 hours for H c2 4 concentration by a gas chromatograph (see Appendix B).
Analysis of variance was performed using SAS to test for statistically significant differences between the treatments. The
Least Significant Difference (LSD) at the 5 percent level for differences between mean values was applied when F-tests were significant. 14
RESULTS AND DISCUSSION
Results from earlier experiments (see Appendix A) had shown that
PEG concentrations of 100, 200, and 225 g/1 gave essentially the same response as the control (0 g/1) and did not cause appreciable abscission. Results from this experiment show PEG concentrations above 225 g/1 caused abscission beginning 72 hours after the experiment began (Table 1). After 120 hours, clear leaf-abscission patterns had developed. The higher PEG concentrations resulted in larger percentages of leaf abscission, up to 70 percent with the 400- g/1 treatment. Statistically significant differences are evident between the 0- and 300-g/1 levels and between the 300-g/1 and 350- or
400-g/1 levels (Table 1).
Stress symptoms were visible after 24 hours of PEG exposure, and plants in the 350- and 400-g/1 groups remained wilted throughout the experiment. Older leaves were the first to abscise and newly expanded foliage tended to abscise only in response to the two most stressful
PEG levels. All abscised leaves were green or only slightly chlorotic at the time of separation from the plant.
Leaves of plants exposed to PEG-induced water stress contained increased internal H within 24 hours and all PEG treatments c2 4 resulted in substantial increases in internal H as compared with c2 4 the control (Figure 1). Statistically, there were no significant differences at the 5 percent level until hour 96. At 96 and 120 hours, the plants in both the 400-g/1 and the 350-g/1 treatments 15
Table 1. Percent leaf abscission over time after osmotically induced water stress
Percent Leaf Abscission
PEG Time from start of PEG exposure concentration (hours) (g/1) 24 118 72 96 120 144 168
1 0 0 0 1a 2a 2a 2a 2a
300 0 0 15b 23b 28b 31b 34b
350 0 0 24b 45b 57c 63c 68c
400 0 0 22b 57c 70c 79c 84c
LSD 12 18 19 19 20 (0.05)
1Means within columns followed by different letters are significantly different from each other at the 5 percent level of LSD (Least Significant Difference).
differed from the control but were not different from the 300-g/1 treatment. In addition, at 96 and 120 hours, the 300-g/1 treatment was not different from any of the other treatments. Although the data from the treatments in Figure 1 appear to be quite different, there were few statistical differences because of large variability within any PEG treatment. As in the leaf-drop study, older leaves abscised first. A key observation from this experiment was leaf abscission did not commence until endogenous H levels reached 1.5 to 2.5 pl/1. c2 4 6 0 !fEG/1--- ...... ,, D)~/t----- ...... / ' ... ,,', ...... ,, ,,'' 350 !fEG/1 ...... '· ,,.. ,... 5 ...... ,;ot~ 400 !fEG/1 ---·.. ··-·- ...... ,.. .. CD - / ,/_.. / ' ,.... / ,, .. i ..... ,, '" / / ' ::! " / ...... ' ' 0 / / . -b r _...,.t:------_..,. ~-/" ' !.. 3 .. ,, - , // / _.,.. / -- ..-....· ,~.,.. -- T ~ //./ ... -- LIJ / / ..... / --·· --- .... ~ 2 -- 0\ X , ...... '!/ ,,,,' LIJ 7 ...... ,~ ,,,,' ...... // ,,.. ., .... 1 .... ,, ...... /''' .:;;:;--~P-=-- ...... ------_..
00 10 20 30 40 50 60 70 BO 90 100 110 120 TIME (hours)
Figure 1. Concentrations of endogenous H# over time after osmotically induced water stress. c2 Vertical bars represent LSD at the 5 percent level at times where differences were significant 17
The results of this research add to the evidence that water stress can promote leaf abscission of Ficus benjamina. However, the symptoms associated with water-stress-induced and light-intensity- induced abscission are different. The color of the foliage at the time of abscission is the most obvious difference. Abscission due to water-stress conditions results in foliage that is green at the time of abscission whereas leaves that abscise in response to a change in irradiance tend to turn yellow gradually before abscising and are almost completely yellow at the time they become detached. Thus, not only do fluctuations in different environmental parameters lead to leaf abscission, but the symptoms involved with the abscission they induce also differ.
Increased internal c2H4 levels clearly are associated with Ficus benjamina leaf abscission, but whether ethylene induces leaf abscission or is produced in response to the process still is not understood. Nevertheless, concentrations of this gaseous plant growth regulator began to rise quickly after plants were exposed to the various PEG treatments and initial leaf abscission was associated with levels of 1.5 to 2.5 pl/1 c2H4 in the internal atmosphere of affected foliage.
These studies indicate interior plantscapers should avoid exposing Ficus benjamina to conditions that promote water stress. Dry growing media, especially when combined with rapid air movement, low relative humidity, high irradiance, and high temperatures should be avoided because they promote rapid water loss from the plant. In 18 addition, high soluble-salt levels in the growing medium and prolonged waterlogged conditions may affect water uptake. Avoiding water stress, along with using light-acclimatized plants, should help eliminate the problem of extensive Ficus benjamina defoliation in interior environments. 19
SECTION II. EFFECT OF WATER STRESS ON FICUS BENJAMINA L. LEAF ABSCISSION 20
INTRODUCTION
Ficus benjamina is used frequently in interior landscaping.
Although its graceful habit and lush foliage keep it in demand, the tendency of its foliage to abscise in response to environmental changes has plagued both commercial growers and consumers.
Researchers have shown that changes in irradiance affect the amount of abscission that occurs after plants are shipped from production areas to their destination (12, 14, 26). These studies have led to the commercial practice of light acclimatization which has lessened, but not eliminated, the problem of Ficus benjamina leaf abscission (14). Additional research has shown that leaf abscission can be promoted by subjecting plants to water stress (49, 50, 51, 52).
Increased leaf abscission was observed with plants grown hydroponically when polyethylene glycol (PEG) 6000 was added to nutrient solutions and with plants grown in sand when water was withheld (51).
The purpose of this study was to determine the effect of different degrees of osmotically induced water stress on the leaf abscission of Ficus benjamina. 21
MATERIALS AND METHODS
Twelve to 18 em long terminal cuttings taken from plants which originated from a common parent plant were rooted in perlite for approximately two weeks. Uniformly rooted plants were selected and moved into growth chambers for five weeks of conditioning of the root and shoot systems. All plants were supported by 70 mm DiSPoaDPlugs in
500 ml mason jars filled with full-strength, aerated Hoagland's
Solution (18). Chamber conditions included a 14-hour daylength at 25°
C with an irradiance of approximately 325 ~E/sec/m 2 supplied by incandescent and cool white fluorescent lamps. During dark hours, the temperature was reduced to 20 0 C. Relative humidity was approximately
65 percent. After conditioning, 45 uniform plants were selected and randomly assigned to five treatment groups of equal size.
To determine the effect of osmotically induced water stress on
Ficus benjamina leaf abscission, the root systems of plants were placed into nutrient solution containing either 0, 265, 300, 330, or
350 g/kg of PEG 8000. Each treatment level consisted of nine replicates. Plants remained in this system for 48 hours. After the
48-hour stress treatment, the roots were rinsed with distilled water and returned to nutrient solution containing no PEG. Leaf abscission was monitored daily for one week from the time treatments were applied, and this experiment was performed three independent times.
Solution osmotic potential values were determined for all treatment solutions each time the experiment was performed using the 22
Wescor, Incorporated Model HR-33T Dew Point Microvolt Meter with Model
C-52 Sample Chambers. Each time a solution was tested, five measurements were made. The highest and lowest values were disregarded and the middle three were averaged and recorded.
Analysis of variance was performed using SAS to test for statistical significance between amounts of leaf abscission resulting from the five treatments. The Least Significant Difference (LSD) at the five percent level for differences between mean values was applied when F-tests were significant. 23
RESULTS AND DISCUSSION
The osmotic potentials of the solutions used as treatments in these experiments ranged from -3.3 to -18.5 bars depending upon the treatment (Table 1).
Table 1. Osmotic potential of solutions of polyethylene glycol (PEG) 8000 in Hoagland's Nutrient Solution at 24° C
Solution osmotic potential PEG (bars) treatment (g/kg) Leaf abscission experiment run number
2 3
0 -3.3 -3.3 -3.6 265 -10.7 -12.7 -11.6 300 -12.9 -14.6 -14.7 330 -15.6 -17.9 -17.2 350 -18.0 -18.5 -17.9
Leaf abscission began after plants were exposed to osmotically induced water stress for between 24 and 48 hours (Table 2). By hour
48, up to 4.2 percent of the foliage had abscised from some treatments. The majority of leaf abscission took place within the first 72 hours of the experiment, 24 hours after the stress treatments were removed by returning the plants to the nutrient solution 24 containing no PEG. This pattern was consistent with earlier observations (see Appendix A). By hour 120, up to 49 percent leaf abscission had occurred. Plants typical of each treatment group at 0,
24, 48, 72, 96, and 120 hours after the start of PEG exposure are shown in Figure 1. 25
Table 2. Percent Ficus benjamina leaf abscission after 48-hour exposure to polyethylene glycol (PEG) 8000
PEG Hours after treatment began treatment (g/kg) 24 48 72 96 120
Run Number 1
1 0 0 Ob Oc Oc Oc 265 0 1.2ab 28.2ab 39.5ab 41. 7ab 300 0 1.8a 20.1b 29.0b 29.9b 330 0 1.2ab 30.3ab 38.9ab 39.7ab 350 0 2.8a 39.4a 48.7a 49.0a
Run Number 2
0 0 Ob Ob Oc Oc 265 0 4.1a 32.2a 37 .5ab 40.9ab 300 0 3.4a 29.8a 32.6b 35.6b 330 0 4.2a 30.5a 34.7ab 40.9ab 350 0 4.0a 35.9a 44.1a 48.6a
Run Number 3
0 0 0 Ob Ob Ob 265 0 1. 8 40.1a 40.8a 44.4a 300 0 2.3 39.5a 40.3a 41.6a 330 0 3.4 38.6a 40.5a 41.9a 350 0 3.0 36.9a 40.4a 43.9a
1Means within columns within any one run followed by different letters are significantly different from each other at the 5 percent level of LSD (Least Significant Difference). 29
All plants except those in the control groups began wilting within four hours after the stress treatments were applied. Older and, to a lesser extent, juvenile foliage was the first to abscise.
These observations are consistent with other work which has shown that water stress promotes increased leaf abscission of cotton (38) and
Ficus benjamina (49, 50, 51, 52), and that abscission of older foliage often occurs first. In contrast to Ficus benjamina leaf abscission that results from different irradiance levels, leaves remained green throughout the abscission process. Leaves of plants that have not been light-acclimatized typically turn yellow before separation from the plant.
During all three runs of this experiment, control plants experienced no leaf abscission while, by hour 120, all PEG-treated plants experienced at least 29.9 percent abscission (Table 2). In many cases, the control was significantly different from all PEG treatments, but plants treated with any of the PEG concentrations did not differ from each other. As the PEG concentration increased, the number of plants that failed to survive the treatment increased, and these plants generally had leaves that died but never became detached from the plant. One explanation for this might be that higher concentrations of PEG quickly resulted in destroyed or impaired root and vascular systems of treated plants. This eventually could lead to the death of the plants and would decrease or cease transport of 1- aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene (C H ), from the roots to the upper portions of the plant as 2 4 30 has been demonstrated in water stressed tomato plants (9, 10).
If H is required for leaf abscission, and if the so-called c2 4 11 ACC signal 11 is not reaching the upper portion of the plant, the fact that leaf abscission associated with the more severe PEG-stress treatments often was not significantly different from that associated with less stressful PEG treatments might be a result of equivalent or lower levels of H present in the leaves of those plants. Perhaps c2 4 the inactivity of the vascular system prohibited their survival but, at the same time, kept ACC from reaching the shoot system and evoking c2H4-mediated leaf abscission to the extent that might have been expected.
The plants subjected to lower concentrations of PEG may have shed more foliage than would have been expected (see Appendix A), in part, because ACC was able to reach the shoot, and, subsequently, the leaf blades and petioles. Loss of leaves would serve as an adaptation to such a stress as it would decrease transpiration. Indeed, tropical
plants, including Ficus benjamina, appear to abscise foliage on a
regular basis in response to the wet and dry seasons of their native habitat (40). Logically, the oldest foliage would be the first to abscise as it may be less photosynthetically productive than younger
leaves.
This research is consistent with the results of others who have
shown that Ficus benjamina leaf abscission is promoted by exposure to osmotically induced water stress (49, 50, 51, 52). Water stress also has been shown to cause boll and leaf abscission in cotton (39). Leaf 31 abscission in cotton, like that observed in this study, generally involves the oldest foliage first and the bulk of abscission occurs after water deficits are relieved (30, 39). Although stress was achieved by adding PEG to nutrient solutions, there is evidence that higher molecular weight forms of PEG, such as PEG 8000, are effective at mimicking more realistic conditions including infrequent irrigation of soil-based media (31) without being absorbed by plant tissue (42).
Therefore, commercial growers and consumers should take precautions to prevent conditions promoting high transpiration and water stress.
These would include unusually high temperatures, irradiance, and rates of air circulation, especially when combined with low relative humidity and high soluble salt levels in the root medium. Avoiding water stress and utilizing light acclimatized plants should help eliminate the problem of extensive Ficus benjamina leaf abscission. 32
SECTION III. EFFECT OF WATER STRESS ON ENDOGENOUS ETHYLENE CONCENTRATIONS IN FICUS BENJAMINA L. LEAVES 33
INTRODUCTION
The many ornamental characteristics of Ficus benjamina have made it one of the most frequently used plants in the landscaping of interior environments. When healthy, its unique, weeping habit and attractive, dense, glossy foliage account for its popularity. Often, however, Ficus benjamina are characterized by their tendency to abscise foliage in response to environmental changes. The leaf loss may be sudden or occur over a long period of time. In most cases, the older foliage is first to be lost, but young leaves also may abscise, even before they are expanded fully. Affected foliage may turn yellow before separation from the plant or remain green throughout the abscission process.
Considerable research has been performed in an effort to determine what environmental conditions cause Ficus benjamina leaf abscission and how abscission can be prevented. Light acclimatizing plants during production has been the result of research showing that plants grown under high irradiance are more prone to leaf abscission when moved indoors (14). Further research has explored the role of water stress in Ficus benjamina leaf abscission. Additions of polyethylene glycol (PEG) 6000 to the nutrient solutions in which plants were grown hydroponically and withholding water from plants grown in sand promoted abscission (49, 50, 51, 52). Water stressed plants also have a reduced stem caliper and root and shoot dry weights
(28). In addition, the length of time plants remain in darkness 34 during simulated shipping experiments (12, 56), the type of light they receive following such dark periods (54, 55), nutrition programs (13,
26, 29) and the use of antitranspirants (53) have been shown to affect the quality of Ficus benjamina.
Although leaf abscission of Ficus benjamina remains a concern of growers and consumers and can be affected by many different factors, the question of what regulates the process of abscission internally remains unanswered. Research with water stressed Ficus benjamina has shown that as plant water potentials become more negative, leaf abscisic acid (ABA) content increases, but no correlation between leaf
ABA content and percent leaf abscission was found (52). Studies using other species long have recognized the ability of another plant growth regulator, ethylene (C H ), to induce leaf abscission (6, 15, 16, 21, 2 4 23, 27, 44, 48). H , only one part of the plant's regulatory c2 4 hormonal matrix, is thought to stimulate the production of energy-rich compounds required for the synthesis of enzymes that degrade the walls of cells in abscission zones (4, 19, 46).
This study was designed to examine the endogenous H relations c2 4 in leaves of water stressed Ficus benjamina plants. 35
MATERIALS AND METHODS
Terminal cuttings 12 to 18 em long taken from plants which originated from a common parent plant were rooted in perlite for approximately two weeks. Uniformly rooted cuttings were selected and moved into growth chambers for five weeks of conditioning of the root and shoot systems. All plants were supported by 70 mm DiSPo~ Plugs in individual 500 ml mason jars filled with full-strength, aerated
Hoagland's Solution (18). Chamber conditions included a 14-hour daylength at 25° C with an irradiance of approximately 325 ~E/sec/m 2 supplied by incandescent and cool white fluorescent lamps. During dark hours, the temperature was reduced to 20 0 C. Relative humidity was approximately 65 percent. After conditioning, 45 uniform plants were selected and assigned randomly to five treatment groups of equal size.
To determine the effect of osmotically induced water stress on endogenous c2H4 concentrations of Ficus benjamina leaves, the root systems of plants were placed into nutrient solution that contained either 0, 265, 300, 330, or 350 g/kg PEG 8000. Each of the five treatments consisted of three replicates, each containing three plants.
Solution osmotic potential values were determined for all stress treatments each of the two times this experiment was performed using the Wescor, Incorporated Model HR-33T Dew Point Microvolt Meter. Each time a solution was tested, five measurements were made. The highest 36 and lowest values were disregarded and the mean of the middle three was recorded.
Endogenous H concentrations were measured at the time of c2 4 introduction to stress and after 2, 4, 6, 12, 24, 36, and 48 hours of exposure. Four leaves and their attached petioles were removed from each plant in each treatment at these times. All 12-leaf samples contained leaves of different ages removed from different areas of the plants. The three sets of four leaves from each replicate were grouped and placed into a system designed for the extraction of gas from within plant tissue (7, see Appendix B). After gas samples were collected, they were analyzed using a gas chromatograph (see Appendix
B). Each replicate resulted in one gas sample at each test time.
Thus, for each treatment at each sampling time, three values of endogenous H , one from each replicate, were obtained. c2 4 Analysis of variance was performed using SAS to test for statistically significant differences between the endogenous H c2 4 concentrations resulting from each treatment at each sampling time.
The Least Significant Difference at the 5 percent level for differences between mean values was applied when F-tests were significant. 37
RESULTS AND DISCUSSION
The osmotic potentials of solutions used as treatments in these experiments ranged from -3.2 to -19.5 bars depending upon the experiment and treatment (Table 1).
Table 1. Osmotic potential of solutions of polyethylene glycol (PEG) 8000 in Hoagland's Nutrient Solution at 24° C
Solution osmotic potential PEG (bars) treatment (g/kg) Ethylene experiment run number
2
0 -3.2 -3.7 265 -11.9 -11.4 300 -14.3 -13.6 330 -17.4 -16.1 350 -19.5 -17.5
Leaves of plants subjected to the various water stress treatments during the first run of this experiment contained less than 0.2 ~1/1 endogenous H at the start of the experiment, and the differences c2 4 between the treatments were insignificant (Figure 1). Two hours after the stress treatments were applied, endogenous C H concentrations had 2 4 risen dramatically, and leaves of plants in the 330-, 300-, 350-, 5 .. 0 ~Gikg --- 3. .. 265 gPEG/kg----- 3.0J- ,... .. 300~------J ~ ~lkg ------'f 2.5 r- I I ...,41 L :\' 950 gPEC/kg ·-·-...... \ I llj:, I --f 2.0 0 .. If,:\\ \I tl I I ''- E • I ~ I ..., ...... 'It \'.I I I , , ,., ~.,.,____ ••...- - It I -- \II 1. 5 1 t·. \ ', ' , ·-~&:----...... U·\ I - ...... _...... ~-...... - z ! \ \'I ;' / - ...... ~,, ...... ,.,..•• ·-•• \ ' ...... \II .. !. ··~ , "' ...... "'- ,.., ...... _..... --...-- • ..J I \t \ I / - ...... ,,. ,/ •••• ,..,.. ... •' -- w : ~ ...... ; ... .,...... ,.. > .- f 1 r·.. \' I ...... , ,.,,,,., ...... ------co ~ 1.0 ~ '~ / . 1 . ,_, ...... _...... __, ,.,..,_...... ,.,. \II .: I ,.,,\"'/..····· -...... '.:::1"",.,... ;::------·JI I .... ·.~- •···· ··-·------. __.... __ ...... 1 II. '~------·-----.... .5 r:lt
0.0 I I I I I ' I I I I 0k- 4; 8 12 16 20 24 28 32 36 40 .... 48I TIME (hours)
Figure 1. Endogenous ethylene concentrations in leaves of water-stressed Ficus benjamina over time during run 1. Vertical bars represent LSD at the 5 percent level at times where differences were significant 39
265-, and 0-g/kg groups contained approximately 2.7, 2.3, 1.5, 0.9, and 0.2 ~1/1 endogenous c2H4, respectively. All PEG-treated plants were significantly different from the control at this time except the
265-g/kg group which was significantly different from the 330- and
300-g/kg groups, but not different from the 350-g/kg group.
Concentrations of H in leaves from plants in the 350-g/kg treatment c2 4 were significantly different from those in the 330-g/kg group but not from those in the 300-g/kg group. The difference between the 330-g/kg treatment and the 300-g/kg treatment was not significant.
With the exception of the 265-g/kg treatment, endogenous c2H4 concentrations dropped by hour 4, and all PEG treatments were significantly different from the control but not from each other
(Figure 1). H concentrations in leaves of plants in all treatments c2 4 continued to drop through hour 6, and the statistical significance pattern was identical to that at hour 4.
From hours 6 to 48, the overall trend was for endogenous c2H4 concentrations in the leaves of stress-treated plants to increase, though at hour 24, leaves of plants in all treatments contained less
H than at hour 12 (Figure 1). No significant differences existed c2 4 among any of the treatments at hours 12, 24, and 48. At hour 36, leaves of PEG-treated plants, except those in the 265-g/kg group, contained significantly more H than did leaves of control plants. c2 4 In addition, leaves of plants in the 300-g/kg treatment contained significantly more C H than did leaves of plants in the 265-g/kg 2 4 treatment. 40
During the second run of this experiment, there were statistically significant differences between at least some of the treatments at all times of measurement (Figure 2). At hour 2, plants in the 265-, 350-, and 300-g/kg treatments contained significantly different amounts of endogenous H than the control and the plants c2 4 in the 265-g/kg treatment differed significantly from the 330-g/kg treatment. Endogenous H levels in all treatments except the 265- c2 4 g/kg group continued to rise through hour 4 when leaves of plants in the 265-, 300-, 350-, 330-, and 0-g/kg treatments contained approximately 2.9, 2.9, 2.1, 1.9, and 0.5 ~1/1 c2H4, respectively (Figure 2). At this time, the 265-, 300-, and 350-g/kg treatments were significantly different from the control. Six hours after the stress treatments began, all PEG-treated groups were significantly different from the control even though H levels in all of those c2 4 treatments dropped sharply. In addition, the 265- and the 350-g/kg treatments were significantly different from each other. H c2 4 concentrations in plants of all treatments except 300 g/kg continued to fall through hour 12 when plants in all PEG-treated groups were significantly different from the control but not from each other
(Figure 2).
After 12 hours of exposure, the overall trend was for endogenous
H concentrations to begin again to rise in leaves of plants of all c2 4 osmotic stress treatments. At hours 24 and 36, the pattern of significant differences from hour 12 remained the same except that, at ETHYLENE (mlcrol lter®/I) Figur`e2.Endogenousethyleneconcentr'ationsinleavesofwater'-stressedE±£!±±ben'aminaover` - :T .-- }} }J SIL} .CAJ CJI C] CJI C= Cn C> tln
differ'enceswereslgnificant timedur`ingrun2..Vcr`ticalbarsrepr'esentLSDatthe5per'centlevelattimeswher'e
E TIME(hour®) 2428
32 42 hour 36, the 300-g/kg treatment was not significantly different from the control. At hour 48, endogenous H concentrations in leaves of c2 4 plants in the 330- and 350-g/kg treatments fell and were not significantly different from the control. The H concentration in c2 4 leaves of plants in the 265- and 300-g/kg groups rose from their levels at hour 36, and both were significantly different from the control. In addition, the 265-g/kg treatment was significantly different from the 350-g/kg group (Figure 2).
Although there were differences in H concentrations and c2 4 patterns of significant differences between the two runs of this experiment, the overall pattern of behavior of endogenous H after c2 4 osmotic water stress of Ficus benjamina was consistent. H levels c2 4 rose and declined sharply within the first 6 hours of stress and then increased gradually throughout the remainder of the 48-hour period.
By hour 48, leaf abscission had begun. Thus, between 1.25 and 3.0
~1/1 endogenous H is associated with initial leaf abscission. c2 4 Whether the initial, dramatic rise in H alone is enough to induce c2 4 abscission is not known and should be addressed by further study.
The fact that endogenous H concentrations rise and fall so c2 4 quickly after stress begins may be a result of stomatal condition.
Stomates of other plants almost instantly respond to changes in hydrostatic pressure (57). Perhaps the increase in H is gradual c2 4 throughout the 48-hour period, but stomates closing and reopening within the first six hours of stress might cause a large increase of c2H4 during this time. Furthermore, assuming stomates would react to 43 light and dark cycles while under stress, the c2H4 concentration would be expected to decrease during light periods because open stomates would allow greater gas exchange. This might explain the decrease in endogenous c H at hour 24 in run 1, as this measurement was taken in 2 4 the middle of a light cycle. By hour 48 of run 1, during another light cycle, perhaps the H increase was large enough to mask the c2 4 effect of open stomates or the stress itself caused the guard cells to close, resulting in an increase in H concentration at hour In c2 4 36. addition, the level of abscisic acid, which has been shown to increase in water stressed Ficus benjamina (52), probably would play a role in stomatal behavior.
Water stress has been shown to cause increased H levels in c2 4 leaves of other plants. After seven days of drought stress, endogenous H concentrations in leaves of Vicia faba increased from c2 4 approximately 0.4 ~1/1 to approximately 1.1 ~1/1, and leaf abscission was three times greater with stressed plants than with controls (17).
Additional studies have shown that H production rates rise in c2 4 response to water stress of cotton, and that the degree of water stress linearly correlates with the amount of leaf abscission (38).
Another speculative, yet feasible, explanation for the rapid rise and decline of endogenous H within the first 6 hours of stress c2 4 involves the activity of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of H , in water stressed plants. ACC serves c2 4 as a root-derived signal for increased C H production in the shoot 2 4 system of waterlogged tomato plants (8, 9, 10). During the first few 44 hours, ACC may be acting in the same way in osmotically water stressed
Ficus benjamina. Because the levels of stress were rather severe, the function of the root and xylem systems might have become impaired after the first few hours, preventing ACC transport and contributing to a net reduction in the concentration of endogenous H • c2 4 45
SUMMARY AND CONCLUSIONS
Leaf abscission of Ficus benjamina L., a plant used frequently in interior landscaping, has been a concern of both commercial growers and consumers. The objectives of this research were to determine the effect of water stress on leaf abscission and to examine the relationship between endogenous ethylene (C H ) concentrations and the 2 4 abscission process.
Hydroponically grown Ficus benjamina were subjected to various degrees of water stress by adding different amounts of polyethylene glycol (PEG) 8000 to nutrient solution. After 48 hours of stress, plants were returned to nutrient solution free of PEG and leaf abscission and endogenous H concentrations in leaves were c2 4 monitored.
Early results had shown that PEG added to solutions at concentrations of 225 g/1 or less did not promote abscission or substantial increases in endogenous H levels. PEG added to c2 4 solutions at rates of 265, 300, 330, and 350 g/kg resulted in 29.9 to
49.0 percent abscission 72 hours after the 48-hour stress was relieved, and the amount of abscission was not proportional to PEG concentration. The bulk of abscission occurred 24 hours after stress was relieved, and most leaves remained green throughout the abscission process. Endogenous H concentrations rose and declined c2 4 dramatically in leaves of water-stressed plants within the first six hours of stress and then increased gradually to 1.25 to 3.0 pl/1 at 46 hour 48 when leaf abscission began.
This research has shown that water stress can cause Ficus benjamina leaf abscission. There may be a threshold degree of stress, achieved by adding PEG to nutrient solution at a rate between 225 and
265 g/kg, below which leaf abscission is not promoted. The abscission caused by water stress is symptomatically different from that caused by differentials in irradiance. Increased endogenous c2H4 concentrations are associated with water stress of Ficus benjamina.
Whether water-stress-induced H causes abscission, and, if so, when c2 4 during the course of stress sufficient H is produced to induce c2 4 abscission are questions that should be addressed by future research. 47
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ACKNOWLEDGEMENTS
I wish to thank my major professor, Dr. Richard J. Gladon, for his enthusiasm, guidance, and support. The contributions of the other members of my committee, Dr. Griffith J. Buck and Dr. Nels R. Lersten, also have been appreciated greatly. In addition, I thank the other members of the Department of Horticulture faculty and staff who assisted me throughout my undergraduate and graduate years.
My education has been enhanced greatly through interactions with fellow graduate students in horticulture. Our shared accomplishments and frustrations helped to create friendships that made the ultimate achievement more worthwhile.
Finally, I thank my family for their encouragement and support.
The concern and understanding of my mother has been most meaningful. 53
APPENDIX A. INITIAL WATER STRESS AND ETHYLENE EXPERIMENTATION WITH
FICUS BENJAMINA L.
Before the research included in Sections I, II, and III of this thesis began, experiments involving water stress and ethylene (C H 2 4) in relation to Ficus benjamina leaf abscission were performed. Listed below are pertinent, brief descriptions of portions of this work.
To determine the effect of irrigation regime on Ficus benjamina
L. leaf abscission, sixteen 15 em-potted plants growing in a soilless medium were divided into four treatment groups of equal size. Plants were moved from the greenhouse into a laboratory and placed under cool white fluorescent lamps that were on continuously. Temperature was approximately 24° C with relative humidity of approximately 50 percent. One group was irrigated with tap water as needed, generally twice each week. The media of plants in the second group were kept saturated by irrigations with tap water at least once a day. Growing media of plants in the third group were kept saturated after being allowed to dry until plants were severely wilted, while the containers of plants in the fourth group were submerged completely under distilled water for four weeks. Only plants which were kept saturated after a severe drought stress experienced appreciable leaf abscission.
Leaves of these plants began to abscise 24 hours after the first post- drought saturation. The abscised leaves of plants in the drought stress treatment were collected periodically and analyzed for endogenous H concentration. Between 4.0 and 6.9 ~1/1 of H was c2 4 c2 4 54 found to be present in leaves that had been abscised for no more that
24 hours.
Another experiment involved adding H to the atmosphere c2 4 surrounding 10 em-potted Ficus benjamina plants which were sealed in glass desiccators. After 72 hours, plants held in an atmosphere containing 10 pl/1 H had experienced 52 percent leaf abscission c2 4 while those held in an atmosphere containing 1 pl/1 H had abscised c2 4 no foliage. A similar experiment later showed an atmosphere containing 2 pl/1 H evoked 9.5 percent leaf abscission. c2 4 Initial experiments involving polyethylene glycol (PEG) 8000 additions to nutrient solutions revealed that, when Ficus benjamina were held in either 0, 100, 200, or 400 g/kg PEG for 192 hours, only plants subjected to 400 g/kg PEG experienced uniformly visible stress and substantial increases in endogenous H concentrations. After c2 4 192 hours of continual exposure to stress, plants subjected to 200 g/kg had a maximum of 0.5 pl/1 of internal H while those subjected c2 4 to 400 g/kg PEG contained nearly 4 pl/1 H • c2 4 A subsequent experiment showed plants subjected to stress resulting from addition of 225 g/kg PEG to nutrient solution for 48 hours failed to experience substantial leaf abscission or increases in endogenous H • c2 4 55
APPENDIX B. GAS CHROMATOGRAPH CONDITIONS AND CDS-111 ANALYSIS PROGRAM
Determinations of ethylene (C H ) concentrations were made using 2 4 a Varian 3700 Gas Chromatograph with a flame ionization detector.
Components of gas samples were separated by a 180 em by 1.3 em (6 feet by one-quarter inch) stainless steel column packed with 60 to 80 mesh alumina coated with 2 percent Bis (2-methoxyethoxylethyl) ether. Gas flow rates were 65 ml/minute for helium, 29 ml/minute for hydrogen, and 300 ml/minute for air with the column, detector, and injector temperatures set at 80 0 C, 200 0 C, and 60 0 C, respectively.
A Varian CDS-111 Integrator was used to quantify chromatographic results. Standard samples were analyzed with the CDS-111 programmed as shown in Figure 1. After this calibration, unknown samples were analyzed with the CDS-111 programmed as shown in Figure 2. A typical
CDS-111 output indicating 1.56 pl/1 H at a retention time of 1.47 c2 4 minutes is shown in Figure 3. Peaks with a retention time of approximately 1.18 minutes are presumed to represent ethane.
Each gas sample was extracted by placing leaves into a glass tube sealed at one end with a rubber septum. The other end of the tube was placed vertically into a 9.2 1 glass desiccator containing saturated ammonium sulfate. The solution was drawn into each tube, completely surrounding the leaves, by vacuum force. Once all tubes were in place, the desiccator was sealed and a connected vacuum pump turned on, forcing internal gas out of the leaves. Gas was collected for approximately 2 minutes. The vacuum then was relieved, and the gas 56 which had collected in each tube was drawn into a syringe and analyzed. 57
SECTION 1 1 IDil 4i1 2 S/N 2N 3 IPW 4S 4 TAN% 3.20% 5 AREA 50A 6 STOP 1.80M
SECTION 2 LINE TIME EVT VALUE 1 .00 2FB .50
SECTION 3 1 CALC 4il 2 VIAL 1il 3 SEND Oil 4 LINK Oil 5 DCML 2il 6 UPF .OOOOOOil 7 REF 5% 8 MUST OA 9 MUST O% 10 DATA 5% 11 DATA .OOM 12 FACT 10% 13 ALRM 0/1
SECTION 4 PK TIME AMOUNT AVE.FACT 1 1.47 .940000 .000000
SECTION 5 VIAL SAMP STD/CAL SCALAR 1 1.000000 1.000000*1.000000
Figure 1. CDS-111 program used for injections of standards 58
SECTION 1 1 ID# 4# 2 S/N 2N 3 IPW 4S 4 TAN% 3.20% 5 AREJ 50A 6 STOP 1.80M
SECTION 2 LINE TIME EVT VALUE 1 .00 2FB .50
SECTION 3 1 CALC 4# 2 VIAL 1# 3 SEND 0# 4 LINK 0# 5 DCML 2# 6 UPF .000000# 7 REF 5% 8 MUST OA 9 MUST 0% 10 DATA 5% 11 DATA .OOM 12 FACT 10% 13 ALRM 0#
SECTION 4 PK TIME AMOUNT AVE.FACT 1 1.47 .940000 .491503
SECTION 5 VIAL SAMP STD/CAL SCALAR 1 1.000000 1.000000 1.000000
Figure 2. CDS-111 program used for unknown samples 59
VII 1 INJ. 0 FILE 1 ID# 1 PK * TIME AREA EXT. STD • 50 35812 .oo p .61 33553 .oo 1.18 4376 .oo 1P 1.47 31749 1.56 TOTAL 105490 1. 56 SAMP 1.000000 SCALAR 1.000000
Figure 3. CDS-111 output indicating 1.56 ~1/1 H c2 4