& Hort. Digest 21:71-72. 9. Sharpe, R. H. and W. B. Sherman. 1971.

Breeding blueberries for

6 . Galletta, G. J. 1975. Blueberries and cranberries, p. 154—196. In:
J. Janick and J. N. Moore (eds.) Advances in fruit breeding. Pur­
10.
due Univ. Press, West Lafayette, Ind.
7. Moore, J. N. 1965. Improving highbush blueberries by breeding
and selection. Euphytica 14:39—48.
8 . Morrow, E. B. and George M. Darrow. 1950. The Murphy and 11.
Wolcott blueberry varieties. N. C. Agr. Expt. Sta. Spec. Cir. 10.
low chilling requirement. HortScience 6:145-147.
Sharpe, R. H. and W. B. Sherman. 1976. Flordablue and
Sharpblue, two new blueberries for central Florida. Fla. Agr. Expt.
Sta. Cir. S-240.
Sherman, W. B. and R. H. Sharpe. 1977. ‘Avonblue’ blueberry
HortScience 12:510.

J. Amer. Soc. Hort. Sci. 107(1): 109-112. 1982.

Two Bioassay Techniques for Determining Abscisic

Acid Concentrations1
T . J. Bakken and A. A . Boe2
D epartm ent o f Plant & Soil Sciences, University o f Idaho, M oscow, ID 83843
Additional index words. Plant growth regulators, Phaseolus vulgaris, Lactuca sativa
Abstract. Two bioassay techniques for determining abscisic acid (ABA) concentrations are reported. One used bean
(Phaseolus vulgaris) embryos while the other used lettuce (Lactuca sativa) hypocotyls. The bean embryo assay requires
that seeds first be soaked over night, then the embryos excised and placed in known concentrations of (RS)-ABA for 24
hours, and finally measured. The lettuce hypocotyl assay utilizes seeds incubated for 48 hours, then placed in known con­
centrations of (RS)-ABA for 72 hours and finally measured. A dose-response curve of each bioassay may then be used to
determine unknown concentrations of ABA. The bean embryo test was more rapid, but the lettuce hypocotyl assay was
simpler and more sensitive. Elongation of bean embryos and lettuce hypocotyls was inversely correlated to the log concen­
tration of (RS)-ABA within the range of 0.01 to 100 and 0.001 to 100 |xg/ml, respectively.

Quantitative assay of ABA is a necessary part of growth reg­
ulator research. While physico-chemical methods of ABA esti­
mation are becoming more wide-spread and accurate, bioassay is
still useful (6 ). Bioassays are especially helpful when many sam­
ples must be assayed, and where physico-chemical equipment is
unavailable. Present ABA bioassays, however, are difficult to
use. Some require that additional growth regulators be added to
produce a response (2 ,1 0 ,1 3 ). Others are long and laborious such
as the rice seedling (7), oat mesocotyl ( 8 ), and cotton explant (1)
bioassays. Still others have a limited sensitive range (7, 9, 13).
Milborrow ( 8 ), in his recent review of ABA bioassays, considers
the cereal coleoptile straight growth bioassays (5) the most fre­
quently used ABA bioassays. Cereal coleoptile bioassays, how­
ever, require the addition of growth promoters, strict laboratory
conditions of high humidity, uninterrupted darkness, and
specialized apparatus.
Another weak point of modem bioassays is inadequate data
analysis. Statistical analysis can increase the value of the data ob­
tained from bioassays. This includes such procedures as data
transformation, regression analysis, and establishment of confi­
dence intervals (3).
'Received for publication May 11, 1981. Approved for publication by
the Director of the Idaho Agricultural Experiment Station as Research
paper No. 8177.
The cost of publishing this paper was defrayed in part by the payment
of page charges. Under postal regulations, this paper therefore must be
hereby marked advertisement solely to indicate this fact.
2Research Associate and Professor of Plant Physiology, respectively.
Frankland and Wareing (4) found that inhibitory substances in­
terfered with lettuce hypocotyl extension by gibberellins. The
same bioassay tested ABA concentration with the addition of gib-
berellic acid ( 2 , 1 2 ).
In this paper, we present the experimental and statistical as­
pects of the bean embryo and lettuce hypocotyl bioassays for
ABA.
M aterials and Methods
Bean embryo bioassay. Snap bean seeds (Phaseolus vulgaris
‘Improved Tendergreen’) were soaked in aerated distilled water
for 24 hr. The seed coat was sliced open and the shoot-root axis
(embryo) excised at the cotyledonary node and placed in distilled
water until use. Five embryos were placed in 45 x 12 mm vials
with plastic stoppers perforated in the center to allow air ex­
change. Each vial contained 0.5 ml of W hite’s (15) inorganic salt
solution (pH 5.5) and a 1 x 2.5 cm piece of filter paper treated
with known solutions of synthetic (RS)-ABA. Seven concentra­
tions of (RS)-ABA were tested; 0 .0 1 ,0 .1 ,0 .5 , 1.0, 10, 50 and
100 |xg/ml with 3 replications per treatment and 5 embryos per re­
plication.
Embryos were incubated in darkness at 26 ± 1.5°C to allow
elongation. The vials were placed on a klinostat rotating at 1 rpm
to reduce embryo curvature.
After 24 hr the embryos were removed and measured from root
tip to primary leaf axil to the nearest 0 . 1 mm on a plastic template.
The template was placed on a bacterological colony counter for
ease of measurement. Two sets of known (RS)-ABA solutions
were incubated and measured at different times.
Milborrow ( 8 ) suggested that the line (of the least squares of the
log ABA concentration plotted against plant elongation) which

J. Amer. Soc. Hort. Sci. 107(1): 109-112. 1982. 109

 most accurately described ABA bioassay data was sigmoidal. The
line of regression may be used for estimating concentration from
the observation means. However, to increase precision, transfor­
mations must be made since the further the observations are from
the treatment mean, the more deviation there will be from the line.
The probit procedure eliminates sigmoidal deviation and
homogenizes the residual variance throughout the range of log
(RS)-ABA concentration. The probit formula is a normalizing
transformation used to straighten sigmoidal data ( 1 1 ) and is the in­
verse of the Gausian transformation.
To obtain probit percent inhibition p f growth, a number of
numerical manipulations were performed. Using the control
mean as baseline, C, percent inhibition of growth for each em­
bryo, X, was calculated as 100 (1— X/C). The data was then nor­
malized by the probit procedure. Probit percent inhibition of elon­
gation was plotted against log (RS)-ABA concentration and 90%
confidence limits were calculated. The experiments were ana­
lyzed in a nested analysis of variance as a completely randomized
design having vials nested within treatments and embryos nested
within vials. After probit transformation removed sigmoidal ten­
dencies, the linear regression was solved using the independent
variables as suggested by Hill and Wimble ( 6 ) as were the confi­
dence intervals (14).
Lettuce hypocotyl bioassay. Lettuce seeds, (Lactuca sativa
‘Grand Rapids’) 1978 crop, were placed in (14 x 1.5 cm) Petri
dishes on Whatman No. 3 filter paper moistened with 15 ml of
distilled water and incubated in a growth chamber with continu­
ous light at 24 ± 1.3°C. Cool White fluorescent tubes were placed LOG OF ( R S ) - A B A C0NC. ( u g / m l )
24 cm above the dishes with a light intensity of 63 |xEm~ 2 s ~ 1 at
Fig. 1. Relationship of (RS)-ABA concentration to bean embryo elon­
the level of the dishes. After 2 days the germinated seedlings were gation.
ready for use in the bioassay.
Seedlings were selected for uniform length and state of de­
velopment and placed in Petri dishes (6.0 x 1.5 cm) on Whatman
mean embryo length, and log ± (RS)-ABA concentration in (xg/
No. 1 filter paper moistened with 3 ml of test solution. Three re­
ml was plotted and presented in Fig. 1. (RS)-ABA caused a nega­
plications of each 0 .0 0 1 ,0 .0 1 ,0 .1 , 1.0, 10, 50, and 100 |xg/ml
tively correlated inhibition of bean embryo elongation. Probit
(RS)-ABA were assayed, with 10 plants per replication. Seed­
transformation created a positive relationship (Fig. 2). A positive
lings were incubated for 2 days in darkness at 24 ± 1 .3°C.
slope was produced because probit is based on inhibition rather
After incubation, hypocotyl lengths were measured to the
than stimulation.
nearest 1.0 mm at 10 x magnification. All standard solutions
The accuracy of the bean embryo bioassay was tested by com­
were prepared from synthetic (RS)-ABA. Two sets of standards
paring the slopes of the regression lines of the 2 experiments. Ex­
were incubated and measured at different times and the data sub­
tracts which produce similar responses in test material in all prob­
jected to statistical analysis as described for the bean embryo
ability contain the same substance. Comparison showed the 2 em­
bioassay.
bryo elongation data slopes revealed no significant difference (P
Results and Discussion < .431). Probit transformation (P < .232) did not significantly
improve slope similarity over embryo elongation data.
Bean embryo bioassay. Results of a bioassay trial with (RS)- To test the repeatability of the bioassay, the residual variances
ABA standards are shown in Table 1. The relationship between
from the two experiments were compared. Both embryo elonga­
tion and probit percent inhibition were found to be significantly
different. Probit transformation (P = .0023), did, however, im­
prove variance stability over that of embryo elongation data (P =
Table 1. Relationship of (RS) - ABA concentration to bean embryo
elongation and probit percent inhibition of elongation. .0006).
Lettuce hypocotyl bioassay. The regression of the negative cor­
Mean Mean relation between hypocotyl elongation and (RS)-ABA concentra­
Concn (RS) - ABA em bryo length probit inhibition tion (Table 2, Fig. 3) was similar to that obtained from the bean
(Mg/ml) (mm) (%) embryo bioassay. Probit transformation again produced a positive
100 7.11 az -0.25 a slope and increased linearity (Table 2, Fig. 4).
50 6.97 a -0.23 a Slopes of the lines, representing (RS)-ABA vs. hypocotyl elon­
10 8.50 abc -0.64 b gation for the 2 experiments were tested for equality. The
1 9.56 bed -0.85 be hypocotyl elongation data slopes were not different, (P < .065),
0.5 10.56 edef -1.08 cd
0.1 11.37 def -1.34 d but the probit procedure (P < .08) improved linearity of the sepa­
0.01 12.86 f -1.42 d rate experiments.
Equality of the residual variances of the 2 experiments was then
zMean separation in columns by Duncan’s multiple range test, 5% level. tested. The hypocotyl elongation variance was significantly dif-

110 J. Amer. Soc. Hort. Sci. 107(1):109-112. 1982.

 LOG OF ( R S ) - A B A CONC. ( u g / m l ) LOG OF ( R S ) - A B A CONC. ( u g / m l )
Fig. 2. Relationship of (RS)-ABA concentration to probit transformed Fig. 3. Relationship of (RS)-ABA concentration to lettuce hypocotyl
bean embryo elongation. elongation.

ferent (P = .00004). After probit transformation, the variance

stabilized and was not significantly different (P = .04).
In subsequent experiments it was found that reducing
hypocotyl number from 10 to 5 had no effect on accuracy. There­
fore, twice the number of replications could be run per treatment
with no increase in the number of hypocotyls measured.
The lettuce hypocotyl and bean embryo assays are quantitative,
quite compatible with common plant growth regulator separation
systems. For TLC, eluates of active regions may be used, or if
desired, eluates may be applied to strips of chromatography or fil­
ter paper. This procedure is applicable to column chromatography
and other chromatographic techniques as well. Both bioassays
have wide sensitivity ranges, a 1 0 , 0 0 0 fold range for the bean em­
bryo and a 1 0 0 , 0 0 0 fold range for the lettuce hypocotyl bioassay.
Finally, both are simple to use and capable of accurate results
without the addition of growth promoters.

Table 2. Relationship of (RS) - ABA concentration to lettuce hypocotyl

elongation and probit percent inhibition of elongation.

Mean Mean
Concn (RS) - ABA hypocotyl length probit inhibition
(jug/ml) (mm) (%>

100 3.33 az 0.93 a

50 4.90 b 0.66 b
10 8.93 c 0.05 c
i 11.73 d -0.04 d
0.1 15.30 e - 1.02 e
0.01 17.31 f -1.37 f LOG OF ( R S ) - A B A CONC. ( u g / m l )
0.001 19.17 g -1.44 f
Fig. 4. Relationship of (RS)-AB A concentration to probit transformed
zMean separation by Duncan’s multiple range test, 5% level. lettuce hypocotyl elongation.

J. Amer. Soc. Hort. Sci. 107(1): 109-112. 1982. Ill

 Literature Cited
1. Addicott, F. T ., H. R. Cams, J. L. Lyon, O. E. Smith, and J. L.
McMeans. 1965. Regulateurs naturels de la croissance vegetale.
C. N. R. S. (1964), Paris, p. 687-703.
2. Aspinall, D., L. G. Paleg, and F. T. Addicott. 1967. Abscisin II
and some hormone-regulated plant responses. Austral. J. Biol.
Sci. 20:869-882.
3. Bailiss, K. W. and T. A. Hill. 1971. Biological assays for gib-
berellins. Bot. Rev. 37:437-479.
4. Frank land, B. and P. F. Wareing. 1960. Effect of gibberellic acid
on hypocotyl growth of lettuce seedlings. Nature 185:225-256.
5. Hancock, C. R., H. W. B. Barlow, andH. J. Lacy. 1964. The East
Mailing coleoptile straight growth test method. J. Expt. Bot.
15:166-176.
6. Hill, T. A. and R. W. Wimble. 1969. A note on the precision of es­
timates of gibberellin concentration from regression lines calcu­
lated from bioassay data. Planta 87:20-25.
7. Koshimizu, K., H. Fukui, T. Mitsui, and Y. Ogawa. 1966. Iden­
tity of lupin inhibitor with abscisin II and its biological activity on
growth of rice seedlings. Agr. Biol. Chem. (Tokyo) 30:941-943.
8. Milborrow, B. V. 1978. Abscisic acid. p. 295. In: O. S. Letham,
P. B. Goodwin, and T. J. V. Higgins (eds.) Phytohormones and
related compounds: a comprehensive treatise. Vol. 1, The
biochemistry of phytohormones and related compounds. Elsevier/
North Holland Biomedical Press, the Netherlands.
9. Milborrow, B. V. 1966. The effects of synthetic dl-Dormin (Abs­
cisin II) on the growth of oat mesocotyl. Planta 70:155-171.
10. Milborrow, B. V. 1974. The chemistry and physiology of abscisic
acid. Annu. Rev. Plant Physiol. 25:259.
11. Natrella, M. G. 1963. Experimental statistics. Handb. 91. U. S.
Dept, of Commerce, U. S. Government Printing Office,
Washington, D. C.
12. Robinson, P. M. and P. F. Wareing. 1964. Chemical nature and
biological properties of the inhibitor varying with photoperiod in
Sycamore (SIC) (Acer pseudo-platanus). Physiol. Plant. 17:314—
323.
13. Sivori, E. M., V. Sonvico, and N. O. Fernandez. 1971. Determi­
nation of abscisic acid following Paleg’s method. Plant & Cell
Physiol. 12:993-996.
14. Snedecor, G. W. and W. G. Cochran. 1967. Statistical methods,
6th ed. Iowa State Univ. Press, Ames. p. 159, 160.
15. White, P. R. 1963. The cultivation of animal and plant cells.
Ronald Press, New York. p. 59.

J. A m er. Soc. H ort. Sci. 107(1): 112-115. 1982.

The Effect of Inhibitors of Protein Synthesis on

Ethylene-induced Senescence in Isolated Carnation
Petals1
G. Wulster, J. Sacalis, and H. Janes
D epartm ent o f H orticulture a nd Forestry, C ook College, Rutgers University, N ew Brunswick, N J 08903
Additional index words senescence, ethylene, Dianthus caryophyllus, cycloheximide
Abstract. Exposure of isolated petals of carnation (Dianthus caryophyllus L.) to ethylene resulted in a rapid loss of fresh
weight and “ in-rolling,” both of which were prevented by pretreatment of petals with cycloheximide (CHI). Actinomycin
D (ActD) had no effect on these responses. Treatment of petals with ethylene also resulted in an increase in 0 2 uptake,
which was also inhibited by pretreatement with CHI. “ In-rolling” of petals was prevented only if CHI was applied to pet­
als within 4 hours after beginning the exposure to ethylene, but an 8 hour delay in application of CHI after exposure to eth­
ylene was effective in inhibiting the loss of fresh weight.

Protein synthesis has been shown to play an essential role in the
ethylene-induced ripening of climacteric fruit (3), and the pro­
grammed senescence of leaves (9). The pattern of senescence dis­
played by carnation flower petals resembles that of climacteric
fruits in that both undergo a single sharp transitory increase in
C 0 2 and ethylene production. Carnation petals and ripening fruit
also exhibit similar patterns of protein synthesis, wherein an in­
crease is observed until the respiratory climacteric, followed by a
rapid decline (3 ,4 ).
'Received for publication February 21, 1981. This work, a paper of the
Journal Series, was performed as a part of NJAES Project No. 12143,
supported by the New Jersey Agricultural Experiment Station, Hatch
Funds. Department of Horticulture and Forestry, Cook College, New
Brunswick, NJ 08903.
The cost of publishing this paper was defrayed in part by the payment
of page charges. Under postal regulations, this paper therefore must be
hereby marked advertisement solely to indicate this fact.
Petal “ in-rolling” and a rapid decrease in fresh weight are pa­
rameters which are clearly an indication of the terminal stage of
carnation petal senescence. The onset of this stage can be induced
by ethylene (7). By using these parameters as a reflection of
senescence in isolated carnation petals treated with inhibitors of
protein synthesis, evidence was obtained to support the concept
that protein synthesis is essential to the responses observed in car­
nation petals exposed to ethylene.
M aterials and M ethods
Greenhouse ‘White Sim ’ carnations were grown to provide
freshly-cut flowers which were harvested at a stage when the
outer petals were prependicular to the longitudinal axis of the
flower. Outer petals were removed by cutting at the base near the
junction of the petal and receptacle.
To minimize the possible effect of variability in physiological
states among flowers sampled (6 ), 4 flowers were harvested, and
4 petals from the outer whorl of each were removed in each exper­
iment. In that manner, each petal in a single treatment was from a

112 J. Amer. Soc. Hort. Sci. 107(0:112-115. 1982.