Environmental Monitoring and Assessment (2006) 115: 509–530 DOI: 10.1007/s10661-006-7227-z

c Springer 2006 

A SCREENING TEST FOR THE DETERMINATION OF ETHYLENE SENSITIVITY DANIEL J. ARCHAMBAULT1,∗ , XIAOMEI LI2 , KENNETH R. FOSTER3 and T. R. JACK4 1

Laurentian University, Sudbury, Ontario, Canada; 2 Alberta Research Council Inc. 250 Karl Clark Road, Edmonton, Alberta, Canada; 3 AMEC Earth and Environmental, Calgary, Alberta, Canada; 4 NOVA Chemicals Research & Technology Centre, Calgary, Alberta, Canada (∗ author for correspondence, e-mail: [email protected])

(Received 11 May 2004; accepted 12 May 2005)

Abstract. Ethephon, which releases ethylene within plant tissues after application, was chosen to perform assessments of the relative sensitivity of crops to ethylene and to determine which stages of plant development were most sensitive. The species chosen were: barley, wheat, oats, canola and field pea, all of which are important crops in the province of Alberta, Canada. Plants were treated with ethephon at one of 7 different stages. Plants were assessed for their vegetative and reproductive growth, including height, biomass, yield and seed quality. Visual symptoms were photographed and documented to compare them with symptoms caused by ethylene applied as a gas. It was concluded that in barley, wheat and canola the late vegetative and early reproductive stages were most sensitive, at least when sensitivity was defined as reductions in yield and quality. As for field pea, ethephon had no effect on yield but did cause increased numbers of pods, which in certain conditions could lead to increased yields. Significant effects on vegetative growth were only observed in the early vegetative stages of development but with no effects on yield. The screening protocol successfully identified sensitive cultivars and growth stages for further investigation of the effects of ethylene exposure. Keywords: ethephon, crops, yield, biomass, growth stage, hormone, pollution, air

1. Introduction Ethylene gas (C2 H4 ) has profound effects on a diverse array of plant growth and development processes, including germination, flowering, senescence, abscission, fruit ripening and yield (Abeles et al., 1992). Plants produce ethylene in response to stresses, such as water stress, wounding, or other environmental stresses (Melhorn and Wellburn, 1987; Taylor and Gunderson, 1986; O’Donnell et al., 1996). As a constituent of air, ethylene can also be a phytotoxic pollutant (Squier et al., 1985) with serious consequences primarily in confined areas such as those used for fruit storage. As an indirect or secondary phytotoxicant produced in response to stress agents such as SO2 and O3 , ethylene plays a significant role in the response of plants to air pollutants (Abeles and Heggestad, 1973; Abeles, 1982). In the field, ethylene from anthropogenic sources is thought to be less effective than stressinduced endogenous ethylene because the concentrations are low and exposure

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episodes tend to be transient. However, emissions from point sources such as those from industrial facilities might have negative effects on plants. Fugitive emissions from localized sources in Alberta have the potential to affect the performance of crops. In order to ensure adequate protection of crops from anthropogenic ethylene emissions, the impact of elevated ethylene levels on crop species must first be evaluated. The sensitivity of plant responses to elevated ethylene levels has been shown to be species and growth-stage specific (Abeles et al., 1992). The determination of impacts of ethylene emissions on crops should therefore focus on sensitive species and growth stages. The objective of this study was to determine the sensitivity of selected Alberta crops including an evaluation of differences in sensitivity between growth stages. A total of eight cultivars of five crop species were selected by representatives of industry, crop producers and government for the present study. These crop species and cultivars are considered important agricultural crops in Alberta now and in the future. Limitations imposed by time and space restricted the number of crop species/cultivars that could be studied in detail using an exposure system consisting of six 1.3 m3 Conviron growth-chambers constructed at the Alberta Research Council facility in Vegreville, Alberta (Li and Archambault, 1999). Therefore, the decision was made to focus on species and growth stages that are believed to be most sensitive. Ethephon (2-chloroethylphosphonic acid, Cl CH2 CH2 PO3 H2 ) was used as an ethylene surrogate to perform quick assessments of the sensitivity of the selected cultivars and to determine which stages of plant development were most sensitive. While ethylene (CH2 CH2 ) is a gas, ethephon is regarded as “liquid” ethylene. Ethephon is applied to plants in the form of a mist or a spray. After application, ethephon penetrates through stomata and cuticles to the apoplast where, at pHs of 5.0 and above, it decomposes to form ethylene, chloride and phosphonate in a non-biological, pH-dependent manner according to the reaction: Cl CH2 CH2 PO3 H2 + OH− → CH2 CH2 + Cl− + H3 PO4 . Studies have shown that in barley, ethephon-released ethylene began appearing in tissues after a short lag period and peaked after approximately 8 hours following application (Foster et al., 1992). Because of its liquid form and the path of absorption, plant uptake of ethylene from ethephon differs from exposure to ethylene in the gaseous form. An application of ethephon cannot be equated to a particular exposure (concentration, duration) to ethylene gas. Nevertheless, consistent applications of ethephon may be used to identify relatively sensitive plant species and growth stages for further work without knowing an exact ethyleneequivalent dose. The use of ethephon is therefore regarded as a relatively quick and inexpensive way of assessing the relative sensitivity of plants to exogenous ethylene.

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Having taken the above considerations into account, experiments were devised to identify ethylene-sensitive stages of plant growth in several cultivars of selected agricultural crops. The hypothesis tested was that: plant responses to ethephon treatments will depend on the plant species used and the time of application of the treatment as it relates to the plant’s growth stage. This paper describes the procedures used and the results of these experiments. 2. Materials and Methods 2.1. SELECTION

OF CROP SPECIES

Five crop species were selected to represent cereals, oilseeds and legumes commonly grown in proximity of anthropogenic ethylene sources. The selected species and cultivars were: a) barley (Hordeum vulgare) cvs. AC Lacombe (6 row) and Harrington (2 row), b) wheat (Triticum aestivum) cvs. CDC Teal and AC Taber, c) oats (Avena sativa) cv. Derby, d) canola (Brassica sp.) cvs. Quantum (B. napus) and Reward (B. rapa), and e) field pea (Pisum sativum) cv. Carrera. 2.2. G ROWTH

CONDITIONS AND EXPERIMENTAL DESIGN

2.2.1. The Growth Medium All plants were grown in greenhouses in 20 cm (diameter) plastic pots containing 4 kg of a standard greenhouse soil mixture consisting of equal volumes of sand, soil, peat and vermiculite. The growth medium was initially fertilized with 1.6 g dolomitic limestone (52% CaCO3 and 41% MgCO3 ) and 1.0 g superphosphate (45% available P2 O5 ) per pot (ARC standard procedure). Following emergence, the growth medium was fertilized on a weekly basis using NH4 NO3 and/or KH2 PO4 to maintain total available N at approximately 300 ppm and P at approximately 100 ppm. In the case of canola, sulfur was added in a 1:6 ratio with nitrogen (1S:6N). Micronutrients were also added periodically using a 1/4 strength modified Hoagland’s solution containing only micronutrients. Three days prior to fertilizing, six to ten soil samples were taken from randomly selected pots from each experiment and analyzed for available N and P (Yeung, 1992). 2.2.2. Growth Conditions Light, temperature and relative humidity were monitored using quantum sensors (Li-190SA, LI-COR Corp.) and temperature/humidity probes (HMP35A, Campbell Scientific). A photoperiod of 16 hours light and 8 hours dark was maintained for all experiments using natural light supplemented by sodium halide lights. Light intensities at mid-canopy were determined to vary between 175 and 795 μmols m−2 s−1 . Temperatures ranged from 20 to 32 ◦ C in the daytime and 14 to 20 ◦ C at night. Relative humidity ranged from 10 to 70%. In all experiments, plant densities were 3 per pot for cereals and canola and 4 per pot for field pea.

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2.2.3. Experimental Design The experimental design for each experiment was: 1 cultivar × 4 replicates × 8 treatments (including controls) = 32 pots total, arranged in a completely randomized design. To minimize the effects of heterogeneous conditions within the greenhouses pots were re-randomized to new positions within the greenhouses on a bi-weekly basis following emergence. Re-randomization was ended when the plants became most vulnerable to mechanical damage (mostly due to physical size). These stages were: boot swelling for cereals, many flowers open for canola and flat pod for field pea. 2.3. E THEPHON

APPLICATIONS

2.3.1. Growth Stages for Ethephon Treatment Plants were treated with ethephon at one of 7 different stages depending on plant type. Four replicates were used for each of the treatment stages and four pots were designated as controls receiving no ethephon. Experiments contained a total of 32 pots. Stage labels, stage codes and stage descriptions are given in Table I. 2.3.2. Procedure for the Application of Ethephon An application rate of approximately 200 L ha−1 at a concentration of 40 mM was chosen based on the results of preliminary studies. In these studies, a rate slightly above the highest recommended rate of application for agricultural applications (100 L ha−1 , 40 mM) failed to produce effects in barley cv. Harrington, the first test cultivar. Doubling this rate of application produced effects and therefore the double rate was adopted for all formal trials. Ethephon applications were performed using either an automated spraying chamber (stages A and B for cereals, A through C for canola and all stages for pea), or a hand-held paint sprayer (remaining stages). The paint sprayer was used to apply ethephon at growth stages where application from the sides was required to achieve a uniform coverage of the plants and/or when plants were too large to fit in the spray chamber. The rate of application achieved using the spray chamber was converted into mass of ethephon per leaf area. The appropriate rate of application using the paint sprayer was calculated as an extrapolation of the rate achieved using the spray chamber. In all cases, treatment was applied when at least half of the plants within each pot were at the predetermined stages. 2.3.3. Testing for Acidity Effects of Ethephon The pH of ethephon solution applied to plants in these experiments was 1.8. Experiments were conducted using both cultivars of wheat to eliminate the possibility that effects of ethephon might be caused by the acidity of the solution. Ethephon solution was prepared for experimentation and the solution was adjusted to pH 5.0 using 5 M KOH. The solution was stirred under vacuum for 72 hours to

early leaf two (12)

second true leaf formed (2.1) first true leaf expanded – 1 node (101)

Cereals

Canola

flag leaf just visible (37)

lower buds yellowing (3.3) first buds visible (201)

inflorescence visible at center of rosette (3.1)

fifth true leaf expanded – 5 nodes (105)

C

main axis and two tillers (22)

B boots just visibly swollen (43) first flower opening on terminal bud (4.1) first flower open (203)

D

90–100% of pods on main stem full – pods yellowing (208–209)

seeds in lower pods full size (5.1)

flowering complete (4.4) 10–50% of pods on main stem full (207–208)

early milk(73)

beginning of anthesis (61)

first spike of inflorescence just visible (51) many flowers opened (4.2)

pods flat (205)

G

F

E

Note. For cereals, the numbers in parentheses are stage codes according to Tottman (1987). For canola, the numbers in parentheses are stage codes according to Harper and Berkenkamp (1974) where stages C through G pertain to the main stem. For field pea, the numbers in parentheses are stage codes according to Knott (1987).

Field Pea

A

Crop

Stage label

TABLE I Stage label, stage codes and stage descriptions designating the time of ethephon application for barley, wheat and oats (cereals), canola and field pea

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extract the ethylene. Subsequently, the pH of the solution was readjusted to pH 1.8 using 1 M HCl. The solution was applied to wheat plants as an acidity control. Plants were assessed and harvested in the same manner as described for other treatments. Statistical analyses showed no significant effects of acidity on any of the parameters measured, therefore, acidity controls were not used in subsequent experiments. 2.4. M EASUREMENTS

AND ASSESSMENTS

All biomass measurements are on a dry weight basis on samples oven dried at 65 ◦ C for at least 48 hours. 2.4.1. Cereals Vegetative characteristics: • Height of the tallest plant in each pot was measured at harvest. • Total vegetative above-ground biomass was determined. • Length of the second internode and stem thickness at the center of the same internode were measured for all stems bearing inflorescences on one randomlyselected plant per pot. • The number of tillers was counted on one randomly-selected plant per pot at several occasions after treatment throughout the experiment and tiller numbers are reported for counts taken at the early milk stage when tillering appeared complete. (Tiller counts were not taken in this fashion for barley cv. AC Lacombe. This is because these plants lodged and it was necessary to place them in tomato cages to prevent damage. These problems made it too difficult to obtain tiller counts for individual plants within pots). • Total root biomass per pot was determined for barley cv. Harrington, wheat cv. AC Taber and oats cv. Derby. • Root to shoot ratios were calculated. Reproductive characteristics: • • • •

Total numbers and weights of all heads per pot were determined. Total numbers and weights of all seeds per pot were determined. Weight per thousand seeds was calculated. Ground seed samples were analyzed for total nitrogen using the TKN (Total Kjeldahl Nitrogen) method in the Department of Renewable Resources at the University of Alberta in Edmonton, Alberta (McGill and Figueiredo, 1993). • Ground seed samples were analyzed for caloric content per gram of material using Bomb Calorimetry (ASTM Standards for Bomb Calorimetry and Combustion Methods, 1972) in the Department of Agricultural, Food and Nutritional Science at the University of Alberta in Edmonton, Alberta.

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2.4.2. Canola For cv. Quantum Vegetative characteristics: • Height of the tallest plant in each pot was measured at harvest. • Total vegetative above-ground biomass was determined. • Stem thickness was measured at a height of 6–8 cm above ground level on all plants. • Total root biomass per pot was determined. • Root to shoot ratios were calculated. Reproductive characteristics: • Total numbers and weights of all seeds per pot were determined. • Weight per thousand seeds was calculated. • Total numbers and weights of all pods of one randomly-selected plant per pot were determined. • Total numbers and weights of pods per pot were also determined by extrapolating the data obtained for one plant per pot. This was done because it was not feasible to obtain detailed data on a per pot basis because of the large number of pods and seeds per pot. • Samples of whole seeds were analyzed for total oil, protein, calcium and phosphorus content using NIR calibrated against samples analyzed using wet chemistry methods at the Soil and Crop Diagnosis Centre of Alberta Agriculture in Edmonton, Alberta. For cv. Reward • Height of the tallest plant in each pot was measured at harvest. • Total above-ground biomass was determined. • Stem thickness was measured at a height of 6–8 cm above ground level on all plants. • Reproductive characteristics were not determined because uneven pollination (cv. Reward is an obligate out-crosser) would have produced unreliable yield. 2.4.3. Field Pea Vegetative characteristics: • • • •

Height of the tallest plant in each pot was measured at harvest. Total vegetative above-ground biomass was determined. Stem thickness was measured at a height of 6–8 cm above ground level. Total number of leaves was determined for one randomly-selected plant per pot.

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• Total root biomass per pot was determined. • Root to shoot ratios were calculated. Reproductive characteristics: • • • • •

Total numbers and weights of all pods per pot were determined. Total numbers and weights of all seeds per pot were determined. Numbers of yellow and green pods per pot were counted. Weight per thousand seeds was determined. Ground seed samples were analyzed for total nitrogen using the TKN method in the Department of Renewable Resources at the University of Alberta in Edmonton, Alberta. (McGill and Figueiredo, 1993). • Ground seed samples were analyzed for caloric content per gram of material using Bomb Calorimetry (ASTM Standards for Bomb Calorimetry and Combustion Methods, 1972) in the Department of Agricultural, Food and Nutritional Science at the University of Alberta in Edmonton, Alberta. 2.5. D ATA

ANALYSIS

Treatment effects were computed using an ANOVA and differences between treatments were determined using Duncan’s Multiple Range tests. A one-way ANOVA was conducted in which the effects of application of ethephon at different growth stages on vegetative and reproductive characteristics were compared.

3. Results 3.1. B ARLEY ( CVS .

HARRINGTON AND AC LACOMBE )

3.1.1. Vegetative Characteristics Although plant height was reduced soon after treatment with ethephon at the twoleaf and two-tiller stages for both cultivars for a few weeks (data not shown), no significant differences in height between treatments and controls were detectable at final harvest in cv. Harrington and approximately 16–17% increases in height were noted in cv. AC Lacombe treated at the early anthesis and early milk stages (Table II), respectively. Results of total vegetative above-ground biomass measurements showed no significant effects of ethephon in cv. AC Lacombe, but 32–38% increases in cv. Harrington when plants were treated at the flag leaf and boot swelling stages (Table III), respectively. Neither of the cultivars exhibited differences in the length of the second internode in response to ethephon. Cultivar AC Lacombe showed a 33% increase in thickness of the main stem when plants were treated at the spike emerging stage (Table II) but no other effects were noted in that cultivar nor in cv. Harrington (Table III). While large variations in tiller numbers were observed in cv.

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TABLE II Effects of the application of ethephon at seven different stages on height (cm), thickness of main stem (mm), weight of heads per pot (g DW) and caloric content of seeds (g−1 DW) in barley cv. AC Lacombe Height

Thickness of main stem Weight of heads/pot Caloric content of seeds

Stage

mean se

stat mean se

stat

mean se

stat

mean se

Control A B C D E F G

56.4 58.4 48.6 52.5 58.4 60.7 66.0 66.0

ab ab d ad ab bc c c

ab abc ab a ab c bc ab

19.7 17.2 11.1 9.8 12.5 18.6 18.7 19.3

a ac b b bc a a a

4387 25 a 4406 40 a 4418 13 a 3683 423 b 4405 28 a 4296 37 a 4340 43 a 4389 28 a

0.9 3.1 2.6 0.4 1.7 2.4 1.4 4.3

2.77 3.22 2.93 2.72 2.94 3.67 3.35 2.95

0.19 0.16 0.14 0.21 0.30 0.26 0.14 0.08

1.5 2.4 0.8 1.3 1.3 3.0 3.5 1.8

stat

Note. Values are means of 4 replicates ± standard error (se). Within each column, means bearing no letters in common are statistically different at P ≥ 0.05.

Harrington, none of these effects proved to be statistically significant, apparently due to large variation within treatments and also in control plants. Total root weights were also highly variable and no significant effects were detected in cv. Harrington. Root to shoot ratios did not differ significantly from that of the control in cv. Harrington. Root data were not obtained for cv. AC Lacombe. 3.1.2. Reproductive Characteristics Cultivar Harrington produced fewer seed heads when plants were treated at the late vegetative and early reproductive stages with statistically significant decreases in plants treated at the boot swelling, spike emerging and early anthesis stages (Table III). Both cultivars produced smaller heads when treated with ethephon at the two-tiller, flag leaf and boot swelling stages and also at the spike emerging and early anthesis stages for cv. Harrington (Table III). Cultivar AC Lacombe produced lower seed numbers and masses when plants were treated with ethephon at the twotiller and flag leaf stages but these effects were not statistically significant (data not shown). Cultivar Harrington produced significantly fewer seeds when plants were treated at the flag leaf, boot swelling, spike emerging and early anthesis stages and lower total seed weights when treated at these stages in addition to the two-tiller stage (Table III). Decreased nitrogen levels were observed in seeds of plants treated at the early milk stage in cv. Harrington (Table III) but no significant effects were observed for cv. AC Lacombe. Total calories per gram dry weight were depressed in seeds of plants treated at the boot swollen and spike emerging stages in cv. Harrington and at the flag leaf stage in those of cv. AC Lacombe (Tables II and III). Decreases in weight per thousand seeds were observed in seeds of plants treated

2.3 4.8 2.6 5.6 8.8 3.2 3.9 5.2

a ab a b b ab ab a

stat

40.5 41.0 31.3 33.7 36.3 33.2 41.6 40.6

2.2 0.9 0.1 0.8 0.4 2.5 0.5 0.5

ab a d cd cb cd a ab

16 1 1 10 15 17 7 2

se a a ab ab b b b ab

stat

3.49 3.24 3.49 3.41 3.34 3.55 3.65 3.17

0.15 0.08 0.06 0.14 0.09 0.03 0.02 0.05

ab bc ab abc abc ab a c

Nitrogen content of seeds

70 72 52 57 33 37 42 57

mean

Number of heads/pot

7.0 2.6 1.2 0.5 2.9 2.5 3.6 0.1

se a a b bcd d cd bc a

stat

4488 4452 4448 4312 3716 3853 4523 4434

27 20 35 175 371 437 18 21

a a a ab c bc a a

Caloric content of seeds

32.8 33.1 20.7 14.3 6.2 6.7 15.8 35.1

mean

Weight of heads/pot

524 528 454 206 61 82 225 606

mean 56 40 15 74 13 15 57 3

se a a a b b b b a

stat

Number of seeds/pot

21.4 21.6 14.4 6.9 2.2 2.9 9.3 24.4

mean

3.6 2.2 0.5 2.7 0.5 0.2 2.3 0.4

se

a a b cd d cd bc a

stat

Weight of seeds/pot

Note. Values are means of 4 replicates ± standard error (se). Within each column, means bearing no letters in common are statistically different at P ≥ 0.05.

Control A B C D E F G

73.0 85.5 70.3 93.8 94.0 84.0 83.5 71.8

Control A B C D E F G

se

Weight/1000 seeds

mean

Stage

Veg. a–g biomass/pot

Barley cv. Harrington

TABLE III Effects of the application of ethephon at seven different stages on vegetative above-ground biomass per pot (g DW), number of heads per pot, weight of heads per pot (g DW), number of seeds per pot, weight of seeds per pot (g DW), weight per thousand seeds (g DW), nitrogen content (% DW) and caloric content of seeds (g−1 DW) in barley cv. Harrington

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at the two-tiller, flag leaf and spike emerging stages in cv. Harrington (Table III) but no significant effects were noted in cv. AC Lacombe. These results suggest that cv. Harrington was more sensitive to ethephon treatment than cv. AC Lacombe and that treatment at the late vegetative and early reproductive stages is most likely to impact seed quality. 3.2. W HEAT ( CVS.

CDC TEAL AND AC TABER )

3.2.1. Vegetative Characteristics As was observed for barley, the height of plants was reduced soon after treatment with ethephon at the two-leaf and two-tiller stages for both cultivars of wheat (data not shown). A 9% increase in height was observed when plants were treated at the early anthesis stage in cv. AC Taber (Table IV). Results of total vegetative TABLE IV Effects of the application of ethephon at seven different stages on vegetative above-ground biomass per pot (g DW), height (cm), number of heads per pot, weight of heads per pot (g DW), weight of seeds per pot (g DW), and nitrogen content of seeds (% DW) in wheat cv. AC Taber Wheat cv. AC Taber Veg. a–g biomass/pot

Height

Number of heads/pot

Stage

mean

se

stat

mean

se

stat

mean

se

stat

Ccontrol A B C D E F G

26.4 28.2 39.5 38.5 32.8 44.2 35.6 39.1

1.2 1.3 4.2 3.9 1.9 5.1 1.3 3.2

a a bc bc ab c abc bc

51.0 54.0 57.0 53.2 50.9 52.2 60.4 57.3

5.2 4.6 6.6 3.8 3.0 3.2 1.7 9.5

a ab ab ab a ab b ab

23 30 37 39 38 43 30 38

1 4 8 7 3 6 5 4

a ab ab ab ab b ab ab

Weight of heads/pot Control A B C D E F G

17.0 21.7 24.0 18.0 22.5 26.5 22.6 27.4

1.9 2.0 1.4 0.8 1.3 3.0 1.9 2.0

a ab ab a ab b ab b

Weight of seeds/pot 9.6 12.5 13.2 8.1 11.6 15.5 13.5 17.5

1.8 0.9 0.6 0.5 1.3 4.5 2.6 2.0

ab abc abc a abc bc abc c

Nitrogen content of seeds 4.07 3.86 3.79 4.13 3.86 4.10 3.91 3.86

0.17 0.26 0.10 0.16 0.19 0.28 0.26 0.14

ab abc bc a ab abc abc c

Note. Values are means of 4 replicates ± standard error (se). Within each column, means bearing no letters in common are statistically different at P ≥ 0.05.

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above-ground biomass measurements showed no significant effects of ethephon in cv. CDC Teal. While a general trend towards increased vegetative above-ground biomass was noted in all treatments for cv. AC Taber, only those effects at the twotiller, flag leaf, spike emerging and early milk stages proved to be statistically significant with 50, 45, 70 and 50% increases in biomass respectively (Table IV). Neither of the cultivars exhibited differences in the length of the second internode nor in the thickness of the main stem in response to ethephon. Similarly to barley, large variations in tiller numbers were noted but this effect proved to be statistically insignificant, apparently due to large variation within treatments and also in control plants. Total root weights showed high variability within treatments and no significant treatment effects were observed in cv. CDC Teal. As well, no significant treatment effects on root to shoot ratios were observed. Root data were not obtained for cv. AC Taber. 3.2.2. Reproductive Characteristics Cultivar CDC Teal showed a trend towards more heads produced when plants were treated at the early reproductive stages (flag leaf and boots swollen) but none of the treatment effects were statistically significant. In cv. AC Taber, all treatments tended to cause increases in the production of heads, however, statistically significant effects were only observed when plants were treated at the spike emerging stage (Table IV). No treatment effects on total weight of heads per pot were found for cv. CDC Teal, whereas in cv. AC Taber treatment of plants at the spike emerging and early milk stages caused increased head weights of 55 and 60%, respectively (Table IV). In cv. CDC Teal, total numbers and weights of seeds per pot were not significantly affected by treatment with ethephon. In cv. AC Taber, however, seed weights per pot were significantly increased when plants were treated at the early milk stage (Table IV). While no effects of ethephon on nitrogen levels were observed in seeds of cv. CDC Teal, seeds of plants treated at the early milk stage showed depressed nitrogen levels in cv. AC Taber (Table IV). No ethephon effects on total calories per dry weight nor weight per thousand seeds were observed in either of the wheat cultivars. Fewer effects of ethephon on seed quality were observed in wheat than in the barley cultivars. Cultivar AC Taber proved to be more sensitive to ethephon than cv. CDC Teal. 3.3. O ATS ( CV.

DERBY )

3.3.1. Vegetative Characteristics Plant height was reduced by treatment with ethephon at the flag leaf stage (Table V) while biomass and tiller numbers were unaffected by treatment with ethephon. Stem thickness increased significantly when plants were treated with ethephon at the boot swelling stage (Table V). Root weights were significantly greater in plants treated with ethephon at the early anthesis stage (Table V). Root to shoot ratios were not significantly different for any of the treatments.

5.7 4.1 2.6 4.1 3.6 4.3 4.5 5.2

a a a b a a a a

stat

28.5 32.5 32.4 14.8 25.9 34.1 30.0 28.4

4.4 1.7 4.7 4.3 2.4 6.1 4.9 4.9

ab a a b ab a a ab

0.2 0.1 0.1 0.4 0.3 0.2 0.2 0.3

se a a ab ab b ab a a

stat

36.2 39.1 37.7 45.0 34.8 38.0 33.8 37.7

1.3 2.5 2.3 4.3 0.8 0.7 0.6 3.2

a ab ab b a ab a ab

Weight/1000 seeds

4.7 4.7 4.8 4.9 5.5 5.3 4.6 4.7

mean

Thickness of main stem

1.6 1.5 2.5 1.7 1.7 3.0 2.0 1.1

se a a ab ab ab ab b ab

stat

4463 4455 4452 4462 4401 4398 4373 4422

33 19 18 25 9 17 19 17

a a a a ab ab b ab

Caloric content of seeds

12.2 12.2 13.1 16.0 16.2 16.3 19.1 13.9

mean

Weight of roots/pot

2.8 0.6 4.1 4.5 2.9 7.1 4.9 6.8

se a a a b ab a a ab

stat

4.10 4.22 3.88 4.79 3.91 3.92 3.67 3.98

0.30 0.42 0.17 0.57 0.31 0.36 0.41 0.39

a ab a b a a a a

Nitrogen content of seeds

66.8 74.3 74.2 51.2 60.4 70.7 66.3 62.7

mean

Weight of heads/pot

788 848 894 336 742 905 885 772

mean

118 92 178 100 57 174 138 162

se

a a a b a a a a

stat

Number of seeds/pot

Note. Values are means of 4 replicates ± standard error (se). Within each column, means bearing no letters in common are statistically different at P ≥ 0.05.

Control A B C D E F G

111.9 111.0 114.5 93.5 113.3 117.0 112.0 113.0

Control A B C D E F G

se

Weight of seeds/pot

mean

Stage

Height

Oats cv. Derby

TABLE V Effects of the application of ethephon at seven different stages on height (cm), thickness of main stem (mm), weight of roots per pot (g DW), weight of heads per pot (g DW), number of seeds per pot, weight of seeds per pot (g DW), weight per thousand seeds (g DW), caloric content (g−1 DW) and nitrogen content (% DW) of seeds in oats cv. Derby SCREENING TEST FOR THE DETERMINATION OF ETHYLENE SENSITIVITY

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3.3.2. Reproductive Characteristics Total seed weight and number, and total head weight per pot were significantly decreased when plants were treated with ethephon at the flag leaf stage (Table V). The number of heads was also depressed by ethephon at this stage but this effect was not statistically significant. Weight per thousand seeds increased by 20% when plants were treated at the flag leaf stage (Table V). Nitrogen content of seeds was significantly increased (15%) by ethephon application at this stage (Table V) but none of the other treatments affected nitrogen content. Caloric content of seeds was significantly decreased by treatment with ethephon at the early anthesis stage (Table V). 3.4. F IELD

PEA ( CV. CARRERA )

3.4.1. Vegetative Characteristics Heights of the plants were reduced soon after treatment with ethephon at the 1st true leaf and 5th true leaf stages without recovery by final harvest (Table VI). Results of total vegetative above-ground biomass measurements showed a trend towards greater biomass when plants were treated at all reproductive stages (Table VI) with those treated at the first buds visible, first flower, flat pod and pods 10–50% full stages being statistically significant (all ∼50% higher than control). Treatment with ethephon had no significant effect on total number of leaves per plant nor on the thickness of the main stem. Total root weight per pot was increased by treatment with ethephon at all reproductive stages with the exception of the 90–100% pod full stage (Table VI). Because both vegetative above-ground biomass and root weights were increased correspondingly in plants treated with ethephon at the reproductive stages, no significant differences were notable in root to shoot ratios. 3.4.2. Reproductive Characteristics Plants treated at the first buds visible, first flower and flat pod stages exhibited significant increases in total number of pods (Table VI) as a result of a second flush of flowers at later stages in development. These new flowers only produced small flat pods that did not fill or reach maturity by harvest time. This resulted in increases in green pods of 1800% (first buds visible stage), 1300% (first flower stage) and 1400% (flat pod stage) over control plants (Table VI) but no increases in yellow pods and no significant effects on yield as expressed in total numbers and seed weights per pot. A general trend towards decreased weights per thousand seeds was observed when plants were treated at all vegetative stages and at early reproductive stages but a statistically significant difference was only observed in plants treated at the flat pod stage (Table VI). While seed nitrogen levels were depressed when plants were treated with ethephon at the 5th true leaf stage (Table VI), no significant effects of ethephon on calories per dry weight were observed.

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TABLE VI Effects of the application of ethephon at seven different stages on vegetative above-ground biomass (g DW), height (cm), weight of roots per pot (g DW), number of pods per pot, number of green pods per pot, weight per thousand seeds (g DW) and nitrogen content (% DW) of seeds in field pea cv. Carrera Field pea cv. Carrera Veg. a–g biomass/pot

Height

Weight of roots/pot

Number of pods/pot

Stage

mean

se

stat

mean

se

stat

mean

se

stat

mean

se

stat

Control A B C D E F G

9.6 8.6 7.7 14.5 14.2 15.1 14.2 11.4

0.8 1.4 0.3 1.0 1.2 1.9 1.4 1.1

a a a b b b b ab

69.1 51.0 51.8 62.7 72.2 69.1 65.1 68.9

1.0 6.3 0.6 2.7 1.0 2.7 1.9 1.8

ab c c b a ab ab ab

0.92 0.89 0.83 1.46 1.50 1.67 1.69 1.25

0.78 0.12 0.14 0.05 0.07 0.16 0.23 0.24

a a a b b b b ab

19 24 17 47 40 39 27 22

1 3 2 4 4 3 4 2

ab ab a c c c b ab

Number of green pods/pot Control A B C D E F G

2 5 1 28 18 20 8 2

1 3 1 3 4 2 5 2

a a a c b bc a a

Weight/1000 seeds 303.1 289.1 275.5 278.3 279.8 232.7 300.2 309.4

11.1 19.1 5.3 22.9 13.3 17.2 4.4 5.7

a a a a a b a a

Nitrogen content of seeds 3.89 3.92 3.64 3.99 4.08 4.00 3.93 3.75

0.13 0.29 0.09 0.02 0.10 0.09 0.16 0.13

ab ab c a a a ab bc

Note. Values are means of 4 replicates ± standard error (se). Within each column, means bearing no letters in common are statistically different at P ≥ 0.05.

3.5. C ANOLA ( CVS.

QUANTUM AND REWARD )

3.5.1. Vegetative Characteristics In both cvs. Quantum and Reward, treatment with ethephon in early vegetative stages caused severe leaf chlorosis, necrosis, leaf-curl and occasionally leaf abscission. Nonetheless, plants recovered and treatment with ethephon only significantly reduced total plant height at time of harvest when plants of cv. Quantum were treated at the first flower open stage (Table VII). In cv. Quantum, a general trend towards decreases in vegetative above-ground biomass were observed for ethephon treatments at all stages relative to controls. Treatment with ethephon caused significant decreases when plants were treated at the first flowers open stage (45%) and at the many flowers open stages (60%) (Table VII). In cv. Reward, no significant

1.9 2.5 6.3 5.5 7.6 7.3 4.6 4.1

a abc abc abc b abc bc ab

stat

1607 1407 1313 1195 815 872 1103 951

171 153 149 167 150 174 135 181

a ab abc abc c bc abc bc

6.7 3.6 9.0 6.7 8.6 7.0 5.3 10.5

se a ab ab ab b ab ab a

stat

114 95 104 89 93 102 105 103

9 5 12 4 20 5 9 13

ab a abc c bc abc a abc

Number of seeds/pot (×100)

169.8 159.0 158.1 159.8 143.8 151.6 159.0 168.9

mean

Height

0.6 0.5 0.5 0.8 0.2 0.4 0.2 0.2

se a ab ab a ab b ab ab

stat

3.13 2.66 3.04 3.10 3.06 3.25 3.29 3.28

0.20 0.11 0.10 0.14 0.12 0.08 0.10 0.14

a b a a a a a a

Weight/1000 seeds

14.3 13.5 12.9 14.4 12.1 11.1 12.6 13.8

mean

Thickness of main stem

1.5 1.2 1.1 1.5 0.9 1.6 1.1 0.9

se a b b ab b ab b b

stat

1.11 1.21 1.13 1.20 1.12 0.96 1.04 1.03

0.04 0.03 0.16 0.03 0.06 0.04 0.01 0.05

ab a ab a ab c bc bc

Phosph. content of seeds

12.9 8.0 8.4 11.6 7.9 10.0 8.0 8.1

mean

Weight of roots/pot

12.5 9.3 8.7 11.5 9.5 10.1 9.4 8.0

mean 1.4 1.3 0.6 0.9 0.6 1.2 1.1 0.7

se

a abc bc ab abc abc abc c

stat

Root to shoot ratio (×100)

Note. Values are means of 4 replicates ± standard error (se). Within each column, means bearing no letters in common are statistically different at P ≥ 0.05.

Control A B C D E F G

102.3 85.6 95.7 99.9 82.5 97.8 84.5 100.9

Control A B C D E F G

se

Number of pods/pot

mean

Stage

Veg. a–g biomass/pot

Canola cv. Quantum

TABLE VII Effects of the application of ethephon at seven different stages on vegetative above-ground biomass (g DW), height (cm), thickness of main stem (mm), weight of roots per pot (g DW), root to shoot ratio, number of pods per pot, number of seeds per pot, weight per thousand seeds (g DW) and phosphorous content (% DW) of seeds in canola cv. Quantum

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change in biomass was observed in any treatment. A significant reduction in stem thickness was observed only when plants of cv. Quantum were treated at the many flowers open stage (Table VII). In cv. Quantum, ethephon caused reductions in total root biomass when plants were treated at the two true leaves, inflorescence visible, first flower open, flowering complete and lower pods full size stages (Table VII). Root to shoot ratios were only significantly decreased when plants of cv. Quantum were treated at the lower pods full size stage and at the seeds in lower pods full stage (Table VII). No root data were obtained for cv. Reward. 3.5.2. Reproductive Characteristics (cv. Quantum Only) The total number of pods per pot was reduced when plants were treated with ethephon at the first flower open stage, the many flowers open stage and the seeds in lower pods full stage where reductions were of 50, 45 and 40%, respectively (Table VII). The total weight of pods per pot, however, was not significantly reduced suggesting that the pods were larger on those plants with fewer pods. The number of seeds per pot was reduced when plants were treated with ethephon at the lower buds yellowing stage (Table VII). Treatment with ethephon had no effect on oil, protein and calcium contents of seeds. Weight per thousand seeds was only depressed when plants were treated at the two true leaves stage (Table VII). Depressed phosphorus levels were observed in seeds of plants treated at the many flowers open stage (Table VII). 4. Discussion 4.1. VEGETATIVE

CHARACTERISTICS

Treatment with ethephon in vegetative stages caused reductions in plant height soon after application in all cereal and canola cultivars but all cultivars recovered and few significant differences in height were notable between control plants and treated plants at time of harvest. These observations are in agreement with those of Rajala and Peltonen–Sainio (2002) who found early reductions in stem lengths of oat, wheat and barley in response to ethephon application, but at maturity no effect was noted. Ethephon caused an increase in height of plants treated at the early anthesis stage in wheat cv. AC Taber (Table IV). In contrast, pea plants treated in vegetative stages experienced reductions in height but did not recover by time of harvest (Table VI). The capacity of ethephon to reduce stem elongation has been documented (Dahnous et al., 1982; Woodrow et al., 1987; Foster et al., 1991) and ethephon has been used for stunting in order to increase hardiness in tomato plants (Woodrow et al., 1987). Few significant differences between treated and control plants in total vegetative above-ground biomass and root mass were observed in cereals. In canola, biomass was reduced when plants were treated in the late reproductive stages (Table VII) with no effects on root mass and in field pea both above-ground and root biomass were increased when plants were treated

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in the reproductive stages (Table VI). These findings for field pea are in contrast to those of Woodrow et al. (1987) who found that dwarfing did not significantly reduce dry weights of tomato plants. In the cereals and pea, no visual symptoms other than height changes were noticed. In canola, however, severe leaf chlorosis, necrosis, leaf-curl and occasionally leaf abscission were observed when plants were treated at the early vegetative stages. Ethephon has been used as a defoliant in cotton (Morgan, 1969). In this respect, the effects of ethephon seen here for canola appear to mimic well-documented effects of ethylene on cotton. While other studies have shown that tillering is often increased in cereals including barley (Foster et al., 1991) and wheat (Poovaiah and Leopold, 1973) when treated with ethephon, no significant effects were found in the present study. Observation of this effect was perhaps confounded by high variability within and between treatments. Foster et al. (1991) also concluded from their studies that effects of ethephon on tillering were dependent on cultivar and on environmental conditions. While Tripathi et al. (2003) observed decreases in length of the third internode of wheat treated with ethephon at a stage approximately equivalent to the flag leaf just visible stage (stage C here), no effects of ethephon on the length of the second internode in cereals were noted in this study. In most treatments, this internode was likely no longer growing at time of treatment so the lack of effect on elongation in those treatments is not surprising. Van Andel and Verkerke (1978) found that ethephon promoted internode elongation in Poa pratensis and inhibited elongation in Triticum sp. These authors noted that these phenomena were species specific and also dependent on the time of application. While lodging in cereals was not measured in the present study, it was noticed that ethephon alleviated lodging in barley cv. AC Lacombe. Similar findings in other cereal cultivars are reported in the literature (Foy and Witt, 1987; Taylor et al., 1991). Overall, the effects of early application of ethephon to cereal and canola plants were poorly retained to time of harvest. Dwarfing of field pea by the application of ethephon at early vegetative stages persisted to time of harvest (Table VI). 4.2. R EPRODUCTIVE

CHARACTERISTICS

Both ethephon-induced yield reductions and increases in barley, wheat and triticale cultivars are reported in the current literature (Dahnous et al., 1982; Rajala and Peltonen-Sainio, 2002; Ramos et al., 1989; Simmons et al., 1988; Tripathi et al., 2004). In this study, seed yield was decreased when plants were treated at the early reproductive stages in all cultivars of cereals, although this effect was not consistently significant. In agreement with the present study, Foster et al. (1992) also found that plants treated with ethephon produced more sterile tillers. Although data on ethephon-induced floret sterility were not obtained in the present study, in some instances reductions in grain yield were observed while the number of heads was not significantly reduced suggesting that not all florets bore seeds. Sterile heads were commonly observed. In wheat cv. AC Taber, an increase in

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seed yield was observed when plants were treated with ethephon at the early milk stage. In canola cv. Quantum, fewer pods formed when plants were treated with ethephon at stages from early flowering to early pod fill (Table VII) and the number of seeds per pot was only adversely affected when plants were treated at the first flowers open and lower buds yellowing stages. A general trend towards lower yields (total seed weight per pot) was also observed in these treatments although these differences were not statistically significant. Yield was not affected by ethephon treatments in field pea. However, a large number of small green pods was produced by plants that were treated at reproductive stages (Table VI). Because it is unlikely that pods produced late in the growing season would mature by harvest time, it is possible that these small green pods would hinder the harvest of mature pods. Nevertheless, the possibility that ethephon or ethylene could be used to increase yields in field pea is interesting. While we are not aware of similar findings for pea in the literature, Woodward and Marshall (1988) have suggested that ethephon may disrupt apical dominance and allow the outgrowth of inhibited lateral buds. These buds might form branches that could flower and form additional pods. 4.3. VARIABILITY

OF ETHEPHON EFFECTS

In the present paper general trends are described in spite of the fact that statistical analyses showed that many of the effects observed were not significant. This is because several trends were commonly found in experiments both with cereals and with canola. For example, depressed yield as expressed in various reproductive parameters was almost always present when plants were treated in the late vegetative and/or early reproductive stages. These treatment stages as described in the methods and materials section of this report tend to be of short duration and overlap between stages was commonly observed within the same pot. For example, in barley several stems may have been in the spike emerging stage while others remained in the boot swelling stage. Having observed variations in the intensity of ethephon effects based on growth stage, it is believed that part of the variability observed in the present study is caused by this overlap in plant stages within a single pot and perhaps even greater over replicated pots. It is also possible that variation in the effects of ethephon within treatments was caused by uneven application of ethephon to plants. This problem is inherent in this type of application and may have caused variation despite attempts at minimizing these effects by adapting ethephon application methods to changes in plant morphology that occur naturally through a plant’s development. For all cereal species used in this study, the method of application of ethephon was changed at the flag leaf stage from an automated spray chamber to a hand-held paint sprayer. While this change was necessary to improve the application of ethephon to plant surfaces, it created the potential for the production of artificial differences in sensitivity to ethephon between early (vegetative) and late

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(reproductive) plant stages. Because a “step” in sensitivity rarely occurred precisely at that stage, perhaps that was not the case. High variability was also observed in control treatments for certain measured characteristics. Such variation may arise from growing plants in heterogeneous environments produced in large greenhouses. While attempts were made to alleviate these effects in early stages of plant growth by re-randomizing the pots within the greenhouses, this may not have been totally overcome. High variability within control treatments may also arise from natural variability in the germplasm of the cultivars used in this study. This however, cannot be measured or ascertained here. It may just be that certain plant characters are less well preserved than others. 5. Conclusions The purposes of this study were to screen several crop cultivars for sensitivity to ethylene and to determine which growth stages are most sensitive. While the effects of ethephon observed in this study may not be attributed solely to the effects of ethylene with certitude, many of the effects are consistent with well-documented effects of ethylene. With this and evidence presented in the literature it was concluded that in barley, wheat and canola the late vegetative and early reproductive stages are most sensitive; at least when sensitivity is equated to reductions in yield and seed weight. As for field pea, ethephon had no effects on yield but did cause increased numbers of pods that in certain conditions could lead to increased yields or could impede the harvest process. Nonetheless, significant effects on growth were only observed when ethephon was applied in the early vegetative stages of development. Based on the findings presented in this report, the following crop species and growth stages are recommended for subsequent growth chamber studies using ethylene: Cereal: Barley cv. Harrington at the spike emerging stage – significant reductions in yield were observed. Barley cv. AC Lacombe, wheat cvs. AC Taber and CDC Teal, and oats cv. Derby were not as sensitive. Oilseed: Canola cv. Quantum at the 2nd true leaf or many flowers open stages – effects on vegetative growth were observed when plants were treated at the early vegetative stages and reductions in yield and seed quality were observed when plants were treated at the flowering stage. This cultivar will be used in growth chamber studies. Canola cv. Reward did not perform well in the greenhouse likely because it is an obligate out-crosser and that greenhouse conditions preclude proper pollination. Legume: Field pea cv. Carrera at the flat pod stage – the production of a flush of small green pods was observed when plants were treated at this stage. This could have negative implications on the harvestability of the crop.

Findings of subsequent growth chamber studies using gaseous ethylene will be reported in future publications.

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Acknowledgments This project was funded by Alberta Environment, Alberta Research Council Inc., NOVA Chemicals Corporation, Union Carbide Canada Inc., Dow Chemical Canada Inc., BP Canada Chemical Company, Shell Chemicals Canada Ltd., Celanese and AT Plastics. Thanks to Alberta Agriculture, Food and Rural Development and Agriculture and Agri-Food Canada for providing technical expertise. The authors gratefully acknowledge the contributions of Paul Sharma and John O’Donovan of the Alberta Research Council Inc. Special thanks to Allan Legge of Biosphere Solutions, David Reid of the University of Calgary, Beverly Hale of the University of Guelph and Page Morgan of Texas A&M University for external critical reviews. Thanks to Leah Matheson, Trish Rattray, Dayna McIntyre, Sharon Pappin-Willianen, Jim Storey and Denise Pewarchuk for their technical support. References Abeles, F. B.: 1982, ‘Ethylene as an Air Pollutant’, Agriculture and Forestry Bulletin, The University of Alberta, 5(1), 4–12. Abeles, F. B., Morgan, P. W. and Saltveit jr., M. E.: 1992, Ethylene in Plant Biology, 2nd edition. Academic Press, Inc, New York, 414 p. Abeles, F. B. and Heggestad, H. E.: 1973, ‘Ethylene: An urban air pollutant’, J. Air Pollut. Control Assoc. 23(5), 517–521. ASTM Standards for Bomb Calorimetry and Combustion Methods: 1972, American Society for Testing and Materials. 1916 Race Street, Philadelphia, Pa. 19103, 8–15. Dahnous, K., Vigue, G. T., Law, A. G., Konsak, C. F. and Miller, D. G.: 1982, ‘Height and yield response of selected wheat, barley, and triticale cultivars to ethephon’, Agron. J. 74, 580–582. Foster, K. R., Reid, D. M. and Taylor, J. S.: 1991, ‘Tillering and yield responses to ethephon in three barley cultivars’, Crop Sci. 31, 130–134. Foster, K. R., Reid, D. M. and Pharis, R. P.: 1992, ‘Ethylene biosynthesis and ethephon metabolism and transport in barley’, Crop Sci. 32, 1345–1352. Foy, C. L. and Witt, H. L.: 1987, ‘Ethephon as an anti-lodging agent for barley’, Bull. Plant Growth Reg. Soc. Am. 15, 8–11. Harper, F. R. and Berkenkamp, B.: 1974, ‘Revised growth-stage key for Brassica campestris and B. napus’, Can. J. Plant Sci. 55, 657–658. Knott, C. M.: 1987, ‘A key for stages of development of the pea (Pisum sativum)’, Ann. Appl. Biol. 111, 233–244. Li, X. and Archambault, D.: 1999, Report I: Design and Performance of ARC’s Ethylene Exposure System. Ethylene/Crop Research Project, Alberta Research Council, Vegreville, Alberta, Canada, 50 p. Melhorn, H. and Wellburn, A. R.: 1987, ‘Stress ethylene formation determines plant sensitivity to ozone’, Nature. 327, 417–418. Morgan, P. W.: 1969, ‘Stimulation of ethylene evolution and abscission in cotton by 2-chloroethene phosphonic acid’, Plant Physiol. 44, 337–341. McGill, W. B. and Figueiredo, C. T.: 1993, ‘Total Nitrogen’, in: M. R. Carter (ed). Soil Sampling and Methods of Analysis, Canadian Society Soil of Science Lewis Publishers, pp. 201–211. O’Donnell, P. J., Calvert, C., Atzorn, R., Wasternack, C., Leyser, H. M. O. and Bowles, D. J.: 1996, ‘Ethylene as a signal mediating the wound response of tomato plants’, Science 274, 1914–1917.

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Poovaiah, B. W. and Leopold, A. C.: 1973, ‘Effects of ethephon on growth in grasses’, Crop Sci. 13, 755–758. Rajala, A. and Peltonen-Sainio, P.: 2002, ‘Timing applications of growth regulators to later spring cereal development at high latitudes’, Agr. Food Sci. Finland 11(3), 233–244. Ramos, J. M., Garcia del Moral, L. F., Molina-Galo, J. L., Slamanca, P. and Roca de Tagores, F.: 1989, ‘Effects of an early application of sulphur or ethephon as foliar spray on the growth and yield of spring barley in a mediterranean environment’, J. Agron. Crop Sci. 163, 129–137. Simmons, S. R., Oelke, E. A., Wiersma, J. V., Lueschen and We Warnes, D. D.: 1988, ‘Spring wheat and barley responses to ethephon’, Agron. J. 80, 829–824. Squier, S. A., Taylor, G. E. Jr., Selvidge, W. J. and Gunderson, C. A.: 1985, ‘Effect of ethylene and related hydrocarbons on carbon assimilation and transpiration in herbaceous and woody species’, Env. Sci. Tec. 19(5), 432–437. Taylor, G. E. and Gunderson, C. A.: 1986, ‘The response of foliar gas exchange to exogenously applied ethylene’, J. Plant Physiol. 82, 653–657. Taylor, J. S., Foster, K. R. and Reid, D. M.: 1991, ‘Ethephon effects on barley in central Alberta’, Can. J. Plant Sci. 71, 983–995. Tottman, D. R.: 1987, ‘The decimal code for the growth stages of cereals, with illustrations’, Ann. Appl. Biol. 110, 441–454. Tripathi, S. C., Sayre, K. D., Kaul, J. N. and Narang, R. S.: 2003, ‘Growth and morphology of spring wheat (Triticum aestivum L.) culms and their association with lodging: Effects of genotypes, N levels and ethephon’, Field Crop. Res. 84(3), 271–290. Tripathi, S. C., Sayre, K. D., Kaul, J. N. and Narang, R. S.: 2004, ‘Lodging behavior and yield potential of spring wheat (Triticum aestivum L.): effects of ethephon and genotypes’, Field Crop. Res. 87(2–3), 207–220. Van Andel, O. M. and Verkerke, D. R.: 1978, ‘Stimulation and inhibition by ethephon of stem and leaf growth of some graminae at different stages of development’, J. Exp. Bot. 29, 639–651. Woodrow, L., Liptay, A. and Grodzinski, B.: 1987, ‘The Effects of CO2 Enrichment and Ethephon Application on the Production of Tomato Transplants’, in: M. S. Reid (ed), Manipulation of Ethylene Responses in Horticulture, Acta Hortic. Vol. 201, pp. 133–140. Woodward, E. J. and Marshall, C.: 1988, ‘Effects of plant growth regulators and nutrient supply on tiller bud outgrowth in barley (Hordeum distichum L.)’, Ann. Bot. 61, 347–354. Yeung, P.: 1992, Methods of Soil Analysis Manual, Soils Branch, Alberta Environmental Centre, Vegreville, Alberta. File no. 2600-CZ2/R1. 43 p.