Plant Physiol. (1992) 99, 111-118
Received for publication November 4, 1991
Accepted December 11, 1991
0032-0889/92/99/011 1/08/$01 .00/0
Induction of Somatic Embryogenesis Using Side Chain and
Ring Modified Forms of Phenoxy Acid Growth Regulators1
David A. Stuart*2 and Carol M. McCall3
Plant Genetics, Inc., 1920 Fifth Street, Davis, California 95616
ABSTRACT
These reports should not be interpreted to mean that 2,4D is the only, or even the preferred, synthetic auxin for
inducing the development of somatic embryos. Several relatively thorough studies have compared the structure and
concentration of various synthetic growth regulators for somatic embryo induction. Kamada and Harada (10) determined that carrot somatic embryogenesis can be induced with
a broad range of growth regulators in the following approximate order of effectiveness: indole-3-acetic acid < a-naphthyleneacetic acid < 4-chlorophenoxyacetic acid < 2,4-D < 2,4,5trichlorophenoxyacetic acid. In another study, Gleddie et al.
(6) found that a-naphthaleneacetic acid is the best auxin for
eggplant somatic embryo induction. In two studies on alfalfa
somatic embryogenesis, it was determined that depending on
the time of exposure and concentration, 50 gM 2,4-D would
promote the best development of embryos (20, 26), although
4-chlorophenoxyacetic acid and picloram were also found to
be effective. Various authors have reported that other synthetic growth regulators, such as picloram or dicamba, are
preferred chemicals for promoting the development of somatic embryos in some species (2, 7). Despite these reports,
2,4-D still remains the most popular synthetic growth regulator in the dicots and monocots for the induction of somatic
The induction of somatic embryo development in cell cultures
of alfalfa (Medicago sativa), celery (Apium graveolens), and lettuce (Lactuca sativa) was compared for 2,4-dichlorophenoxyacetic acid (2,4-D) and various phenoxy acid growth regulators.
Tests using a series of straight chain extensions to the phenoxy
acid side chain indicate that phenoxybutanoic acid is active,
whereas the phenoxypropanoic and phenoxypentanoic analogs
are inactive for the induction of alfalfa embryogenesis. Side
branching on the carbon adjacent to the phenoxy group results
in optically active compounds. Racemic mixtures and the (+)
enantiomers of the compounds are active for alfalfa embryo
induction, whereas the (-) enantiomers are inactive and apparently do not inhibit embryogenesis in any way. Development of
alfalfa embryos, as measured by plantlet formation from individual embryos, is improved by 4-(2,4-dichlorophenoxy)butanoic
acid and with side branching at the carbon adjacent to the
phenoxy group compared with induction with 2,4-D. Similarly,
substituted phenoxy acids also enhance somatic embryo development in celery and lettuce when compared with 2,4-D. These
results are discussed with reference to earlier studies on the
structure activity of various synthetic auxins during cell elongation and with reference to the possible importance of auxin
metabolism on subsequent somatic embryo development.
embryo development.
In an earlier report in which seed storage protein deposition
in alfalfa somatic embryos was investigated, workers in this
laboratory discovered that cultures treated with 50 ,M 2,4-D
gave high numbers of somatic embryos but low expression of
storage protein (20). Embryos treated with 10 AM 2,4-D,
however, expressed between 50- and 100-fold more storage
protein, although the yield of early stage embryos was somewhat lower with this level of growth regulator. This suggested
that perhaps embryo development was inhibited by induction
conditions that include high levels of 2,4-D. In fact, other
studies with carrot somatic embryogenesis have shown that
2,4-D prevents embryo development if it is added to the
regeneration medium (9). We felt that one approach to improve induction and development of more mature somatic
embryos would be to investigate changes in the structure of
phenoxy acid growth regulators during cell culture. We report
here that specific chain extension to the phenoxy acid and
that specific branching of the phenoxy acid side chain improves somatic embryo yield and embryo maturation.
The growth regulator 2,4-D is used extensively in plant
tissue culture to induce somatic embryo formation. This
growth regulator was originally used by Reinert (15) and
Halperin (8) in the first experiments on somatic embryogenesis in carrot. Halperin and Wetherell (9) were the first to
recognize the true importance of 2,4-D in the process of
induction of somatic embryos. A review by Kohlenbach (1 1)
describing the general factors that are necessary to promote
somatic embryogenesis refers to 2,4-D as a preferred synthetic
auxin for the induction of somatic embryo development. A
later review by Evans et al. (3) summarized the literature of
somatic embryogenesis and found that, in 57.7% of the reports, 2,4-D was used in the initial stage of dicot tissue culture
to induce the formation of somatic embryos and in all of the
cases surveyed in which monocot regeneration occurs.
' This work was done at Plant Genetics, Inc., which is now part of
Calgene, Inc., 1920 Fifth Street, Davis, CA 95616.
2
Present address: Hershey Foods Corp., Technical Center, 1025
Reese Avenue, Hershey, PA 17033.
3Present address: Calgene, Inc., 1920 Fifth Street, Davis, CA
MATERIALS AND METHODS
Chemicals
The chemicals used in this report were obtained from the
following sources in the purities noted: 2,4-D (Fig. 1, structure
95616.
111
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112
STUART AND McCALL
I), 95% purity; racemic 2-(2,4-dichlorophenoxy)propanoic
acid [VI], 96% purity; racemic 2-(2,4,5-trichlorophenoxy)propanoic acid [VII], 97% purity; and racemic 2-(4chlorophenoxy)propanoic acid [IX], 95% purity were purchased from Sigma Chemical Co. (St. Louis, MO). 4-(2,4Dichlorophenoxy)butanoic acid [III], 98% purity was purchased from Chem Service (West Chester, PA). (+)-2-(2,4Dichlorophenoxy)propanoic acid [XI], melting point
122.2°C, [a]25D4 = 28. 1 in ethanol was a gift from Prof. K.V.
Thimann, University of California, Santa Cruz. Racemic 2methyl-4-chlorophenoxy)butanoic acid [X] melting point
86.5 to 88.0°C; (+)-2-(2-methyl-4-chlorophenoxy)butanoic
acid [XVII], melting point 74.0 to 75.5°C, [a]l'D = +29.46°
in ethanol; (-)-2-(2-methyl-4-chlorophenoxy)butanoic acid
[XVIII], melting point 74.2 to 75.5°C, [a]19D = 29.610 in
ethanol, 3-(2,4-dichlorophenoxy)propanoic acid [II], melting
point 92.5 to 93.5°C; 5-(2,4-dichlorophenoxy)pentanoic acid
[IV], melting point 69.8 to 71.LC; (+)-2-(2,4,5-trichlorophenoxy)propanoic acid [XIII], melting point 144.5 to 145.5°C,
[a]20D = +49.30 in ethanol; and (-)-2-(2,4,5-trichlorophenoxy)propanoic acid [XIV], melting point 144.0 to 145.6°C,
[a]'8D = 49.60 in ethanol were purchased from R.L. Wain,
Crown Point, Scolton Street, Kent, England. (+)-2-(4-Chlorophenoxy)propanoic acid [XV], melting point 104.0 to
105.0°C, [a]25D = +39.80 in ethanol and (-)-2-(4-chlorophenoxy)propanoic acid [XVI], melting point 104.0 to 105.0°C,
[a]25D = 40.10 were gifts from Prof. V. Tortorella, Dept.
Farmico-Chemico, Universita delgi Studi de Bari, Bari, Italy.
Plant Material
Callus cultures were initiated from petiolar explants of
alfalfa (Medicago sativa L. var Regen S clone RA-3) (27).
This clone is derived from a single cycle selection for regeneration of selfed progeny from var Regen S (1). Plants of
celery (Apium graveolens L. var Calmario) were from HarrisMoran Seed (San Juan Bautista, CA). Seed of lettuce (Lactuca
sativa L. var Vangard) obtained from Asgrow Seed Company
(lot No. WSH 139 3.5) were used in tissue culture
experiments.
Plant Physiol. Vol. 99, 1992
medium will not induce significant embryogenesis if callus is
transferred directly to regeneration medium.
For induction of embryogenesis, cells were subcultured
onto induction medium consisting of SH salts and vitamins,
3% sucrose, 50 ,M 2,4-D, 5 ,uM kinetin, and 0.8% agar. Each
90 x 15 mm plate was inoculated with 1 g fresh weight of
callus. The composition of the induction medium was
changed to test the composition of the auxin-like growth
regulator by deleting the 2,4-D from the induction medium
and substituting the appropriate amount of analog. The synthetic growth regulator was added from a filter-sterilized stock
solution to warm autoclaved medium just before solidification. After 3 d of incubation at 270C, callus was removed and
lightly smashed flat with a stainless steel spatula and transferred to regeneration medium.
Alfalfa somatic embryos were regenerated on SH salts and
vitamins with 10 mm NH4' (total) added as (NH4)2 SO4, 30
mM L-proline, and 3% (w/v) sucrose (see ref. 21 for detailed
medium formulation). Amino acid and NH4' additions to
the medium were made by filter sterilizing the components
and adding them to warm, autoclaved medium just before
solidification. Induced callus was inoculated onto the surface
of 0.8% agar-solidified medium at the rate of 75 mg fresh
weight per 60 x 15 mm Petri plate.
Cultures were incubated for 21 d at 270C under 65 ,uE m2
s-' from cool white fluorescent light. Cultures were analyzed
for embryogenesis by counting green, bipolar structures with
a lOx dissecting microscope. Regeneration treatments were
done with 8 to 10 replicate treatments. Scores for replicates
were averaged ± SE.
Conversion, germination of embryos to plantlets, was effected by transferring 20 randomly picked embryos to halfstrength SH salts and vitamins, 1.5% sucrose, and 0.8% agarsolidified medium poured in 90 x 15 mm Petri plate (20).
Plantlet formation was determined by the presence of a root
and at least one trifoliate leaf per embryo after 28 d of
incubation in the light at 27°C. Typically, 6 to 10 replicate
Petri plates were scored per treatment. The conversion frequency was calculated as the percentage of plantlets formed
from the total number of embryos placed onto conversion
medium.
Alfalfa Cell Culture
The culture of RA-3 followed the general procedures outlined by Walker and Sato (25) for culture initiation, longterm subculture, induction, and regeneration. Briefly, petioles
and callus were grown on SH salts and vitamins (16) containing 3% sucrose, 25 juM a-naphthyleneacetic acid, 10 ,uM kinetin, and solidified in 90 x 15 mm Petri plates with 0.8%
agar. This is referred to as alfalfa maintenance medium.
Cultures were grown at 25°C under low light (5 ,uE m-2 s-')
from cool white fluorescent lights for approximately 21 d.
This constitutes one subculture cycle. At the end of each
subculture, cells were harvested from plates, pressed flat with
a stainless steel spatula, and distributed to new medium at
the rate of 1 g per 90 x 15 mm Petri plate. This subculture
at
4 Abbreviations: [a]'D, the optical rotation of a standard solution
00°C in the specified solvent; SH, Schenk and Hildebrandt.
Celery Cell Culture
Young, inner petioles 7.5 cm in length were harvested from
plants grown in the greenhouse. One- to 5-cm sections were
cut from the petioles and a 1-cm cork borer section were cut
from leaves. Explants were then surface sterilized for 2 to 10
min in 0.525% (v/v) sodium hypochlorite solution at pH 7.0
while being immersed in a Branson model B-220 sonicator.
The hypochlorite solution was poured off and explant sections
were rinsed three times with sterile water. The whitened edges
of the explants were removed aseptically with a scalpel and
discarded.
Callus initiation occurred by placing explants onto SH
mineral salts and vitamins (16) containing 5 ,uM 2,4-D, 0.5
AM kinetin, 3% (w/v) sucrose, 0.8% agar, and 1.0 mM filtersterilized penicillin-G poured into 90 x 15 mm Petri plates.
Explants were incubated in the dark for 3 weeks at 25°C.
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SOMATIC EMBRYOGENESIS WITH ANALOGS OF 2,4-D
Explants were subcultured onto the same medium minus the
penicillin-G and incubated for an additional 3 weeks. The
explants and the callus that formed were removed from the
second medium and were dispersed using a sterile spatula.
The callus tissue was spread over the surface of a 90 x 15
mm Petri plate containing 30 mL of SH salts and vitamins,
2.5 to 5.0 Mm 2,4-D, 0.5 tsM kinetin, 3% (w/v) sucrose, and
0.8% agar. Plates were Parafilm wrapped and incubated as
before for 21 d.
Callus was repeatedly subcultured by removing callus from
the last initiation plate, gently dispersing callus, and inoculating 200 mg of cells onto 30 mL of 0.8% agar-solidified SH
salts and vitamins, 1.0 AM 2,4-D, 0.5 AM kinetin, and 3% (w/
v) sucrose contained within a 90 x 15 mm Petri plate. This
is termed celery maintenance medium but also serves to
induce somatic embryogenesis. Plates were incubated as before for 3 weeks prior to repeating subculture or for regeneration of embryos.
For experiments to investigate the inductive effect of the
test growth regulator, the last subculture medium prior to
regeneration served as the test medium. The structure and
concentration of the growth regulator was varied in 2,4-Dfree celery maintenance medium and was compared with the
standard maintenance medium with 2.25 Mm 2,4-D. Filtersterilized test compounds were added to warm, autoclaved
medium just before solidification. Induction of embryogenesis
occurred over a 21 -d period. Callus was next removed from
the test medium, dispersed, and spread with a spatula on the
surface of an empty Petri plate. One hundred milligrams of
callus was then spread evenly onto the surface of 10 mL of
0.8% (w/v) agar-solidified medium containing SH salts and
vitamins, 5 mM NH4', 3% (w/v) sucrose, and 30 mM Lalanine in a 60 x 15 mm Petri plate incubated for 21 d under
the temperature conditions described above. Embryo yields
were then assessed by microscopically counting somatic embryos formed on the plate after 21 d of incubation.
Lettuce Cell Culture
Seeds of Vanguard 75 lettuce were surface sterilized by
washing with 0.525% sodium hypochlorite for 5 min and
were germinated for 4 d under aseptic conditions. Hypocotyl
sections and cotyledons of these seedlings were dissected and
transferred to Murashige and Skoog (13) salts on 0.8% (w/v)
agar-solidified medium that was modified by deletion of
nitrate and ammonium and the addition of 20 mM L-glutamine. This medium served as the callus initiation, maintenance, and induction medium and contained 3% sucrose plus
3 jM 4-chlorophenoxyacetic acid [V] and 10 AM kinetin.
Callus was subcultured every 21 d and incubated at 24°C
under low light (5 ,uE m-2 s-') from cool white fluorescent
lights. For induction experiments, 4-chlorophenoxyacetic acid
was deleted during the last subculture and substituted with
racemic 2-(2,4-dichlorophenoxy)propanoic acid. Regeneration was achieved by transferring the callus to hormone-free
Murashige and Skoog salts (14) medium containing 3% maltose and 0.8% agar. Regeneration was for 21 d at 24°C with
16 h of cool white fluorescent light per day at 65 MLE m-2 s-',
after which time buds or embryo-like structures developed.
113
RESULTS
Alfalfa Cell Cultures
Substitution of the Acetic Acid with Other Straight Chain
Phenoxy Acids
The first test comparison made was of the side chain
modifications to the 2,4-D phenoxy group. The structures of
2,4-D [I] and its analogs 3-(2,4-dichlorophenoxy)propanoic
acid [II], 4-(2,4-dichlorophenoxy)butanoic acid [III], and 5(2,4-dichlorophenoxy)pentanoic acid [IV] are shown in Figure
1. Each of these analogs was added to the induction medium
of alfalfa cell cultures subsequent to regeneration and the
resulting embryo formation is shown in Table I. In the first
experiment, the inductive activity of 2,4-D was compared
with 4-(2,4-dichlorophenoxy)butanoic acid. The level of embryo induction with 4-(2,4-dichlorophenoxy)butanoic acid
was highest at 50 to 100 Mm, but only one-third of the embryo
yield compared with induction with 50 ,AM 2,4-D. Over the
concentrations tested in experiment 2, neither 3-(2,4-dichlorophenoxy)propanoic acid nor 5-(2,4-dichlorophenoxy)pentanoic acid induced significant somatic embryogenesis compared with 2,4-D.
Branched Side Chain Substitution of the Phenoxy Ring
The next series of chemicals investigated were branched
phenoxy acid derivatives (Fig. 2). Side chain branching leaves
an asymmetric carbon atom adjacent to the phenoxy oxygen
resulting in an optically active compound. The first series of
growth regulators tested were readily available as racemic
mixtures of the (+) and (-) isomers. As control treatments,
2,4-D and 4-methyl-2-chlorophenoxyacetic acid [V] were
compared with racemic mixtures of 2-(2,4-dichlorophenoxy)propanoic acid [VI], 2-(2,4,5-trichlorophenoxy)propanoic acid [VII], and 2-(4-methyl-2-chlorophenoxy)pro-
Q
R2
R
-(CH2)XCOOH
3
X=1; R1 =C1; R2=CI; R 3=H
X=2; R1 =CI; R 2=CI; R 3=H
X=3; R1=C1; R2=CI; R 3=H
X=4; R1 =CI; R 2=C1; R 3=H
X=1; R1 =CH3; R 2=CI; R 3=H
II]
UII]
[III]
[IV]
[V]
Figure 1. The structure of the straight chain phenoxy acids tested
or discussed in this report.
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114
STUART AND McCALL
Table I. The effect of 2,4-D [1], 3-(2,4-Dichlorophenoxy)propanoic
[11], 4-(2,4-Dichlorophenoxy)butanoic [Ill], and 5-(2,4-Dichlorophenoxy)pentanoic Acid [IV] on the Induction of Alfalfa Somatic
Embryos
Each culture was treated identically through subculture at which
time cells were induced for 3 d with SH medium + 3% sucrose + 0.5
AM kinetin + the phenoxy acid growth regulator indicated. Cells were
then plated onto regeneration medium for embryo differentiation.
Embryos were counted at 21 d after plating onto regeneration medium. Data are expressed as the mean ± SE of embryos formed per
plate.
Phenoxy Acid
Concentration
Somatic Embryo
Number
M
Experiment 1
2,4-D
4-(2,4-Dichlorophenoxy)
butanoic acid
Experiment 2
2,4-D
3-(2,4-Dichlorophenoxy)
propanoic acid
5-(2,4-Dichlorophenoxy)
pentanoic acid
50
10
50
100
300
500
200 ± 7
50 ± 3
67 ± 11
61 ±5
34 ± 5
41 ± 3
50
0.5
1.0
10
50
100
300
0.5
1.0
10
50
100
300
227 ± 12
0
0
0
0
0
0
0
0
0
0
0
1 ±0
Plant Physiol. Vol. 99, 1992
into the (+) and (-) isomers were available. The activities of
racemic [VII], (+) [XIII], and (-) 2-(2,4,5-trichlorophenoxy)propanoic acid [XIV] were tested (Table III) and compared with 2,4-D and racemic 2-(2,4-dichlorophenoxy)propanoic acid) for their ability to induce alfalfa somatic embryogenesis (Table III). The (-) enantiomer shows only slight
activity between 10 and 100 AM but does not stimulate the
level of somatic embryogenesis that either the racemic or the
(+) forms of 2-(2,4,5-trichlorophenoxy)propanoic acid do.
The maximum activity of the racemic and the (+) enantiomer
of 2-(2,4,5-trichlorophenoxy)propanoic acid occurs between
10 and 100 Mm also. The racemic and the (+) enantiomer are
of nearly identical activity and induce the same amount of
somatic embryos as racemic 2-(2,4-dichlorophenoxy)propanoic acid). Similar results were also found when racemic
mixtures of 2-(4-chlorophenoxy)propanoic [IX] and 2-(2methyl-4-chlorophenoxy)butanoic acid were compared with
their (+) and (-) enantiomorphs (data not shown).
A summary of the inductive activity of a variety of synthetic
growth regulators for alfalfa somatic embryogenesis is shown
in Table IV. The concentration optimum and active range of
concentrations are also given. The most potent auxin, as
indicated by its low concentration optimum, is 2,4,5-trichlorophenoxyacetic acid. Of intermediate potency is 2,4-D and
many of the other auxins tested in this report. Structures of
low potency are 4-chlorophenoxyacetic acid, racemic 2-(2chlorophenoxy)propanoic acid, and picloram. All of the optically active structures are inactive or show only slight activity
when tested as the (-) form. Structures having an odd number
of carbons between the carboxyl group and the phenyl ring
R2
3-
-0
C- R1
cool-'
R14
panoic acid [VIII]. The results are shown in Table II. The
racemic mixture of 2-(2,4-dichlorophenoxy)propanoic acid is
active at all of the concentrations tested, with the highest
response observed at 100 AM. At this concentration, the
racemic mixture is nearly as active as 2,4-D for induction of
somatic embryogenesis. For racemic 2-(2,4,5-trichlorophenoxy)propanoic acid, the dose response for embryo induction
is maximal between 10 and 50 AM and the yield of embryos
is about the same as with 50 Mm 2,4-D. In experiment 3, a
comparison of 4-methyl-2-chlorophenoxyacetic acid and racemic 2-(4-methyl-2-chlorophenoxy)propanoic acid indicated
that both compounds are maximally active between 50 and
300 Mm and each analog induces fewer somatic embryos than
50 Mm 2,4-D.
Resolved stereoisomers of side chain branched phenoxy
acid growth regulators were compared for the ability to induce
somatic embryogenesis in alfalfa. At 100 yM, (+)-2-(2,4-dichlorophenoxy)propanoic acid [XI] induces 234 ± 16 somatic
embryos per plate compared with 248 ± 8 for 50 Mm 2,4-D
and 223 ± 10 for racemic 2-(2,4-dichlorophenoxy)propanoic
acid [VI]. The (-) enantiomer [X], however, was not available
for testing. Several other phenoxy acids that were resolved
RI = CH3; R2 = Cl; R3 = Cl; R4 = H
Rl = CiA3; R2 = Cl; R3 = Cl; R4 = Cl
VI
VIl
R1 = CH3, R2 = Cii3 R3 = Cl; R4 = I1i
VIII
Ix
x
Rl= Cl3; R2 = ti; R3 = Cl; R4 = l
Rl = CH2CH3; R2 = Ci131R3 = Cl; R4 = ti
R2
R34
R2
R
0-C -COOII
H
14
RH4
-0c
11
(+)-Enan tiomers
xi
1R = Cl R2 = C1; R3 = Cl; R4 = Ii
Rl=CHI3R2=Cl;R3=Cl;R4=Cl
Rl=CHIR2=lI; R3=Cl; R4=H
R1 = Cl 12Ci13
-CR4I
R,
(-)-Enan tioiners
X)ll
xv
xvii
H
XMI
xiv
xvi
R2 = City R3 = Cl; R4 = H xviii
Figure 2. The structure of racemic (top) and optically active (+)
enantiomers (bottom left) and (-) enantiomers (bottom right) of the
branched side chain phenoxy acids tested or discussed in this report.
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115
SOMATIC EMBRYOGENESIS WITH ANALOGS OF 2,4-D
are active for embryogenesis but those having an even
show no activity.
Studies of the Effect of Certain Phenoxy Acid Auxins on
Alfalfa Somatic Embryo Development
Table II. The Effect of 2,4-D [I], 4-Methyl-2-Chlorophenoxyacetic
Acid [VJ, Racemic 2-(2,4-Dichlorophenoxy)propanoic Acid [VI],
Racemic 2-(2,4,5-Trichlorophenoxy)propanoic Acid [VII], and
Racemic 2-(4-Methyl-4-Chlorophenoxy)propanoic Acid [VIII] on the
Induction of Alfalfa Somatic Embryos
Concentration
Phenoxy Acid
In addition to causing the induction of somatic embryos,
the developmental stage and synchrony of embryos is changed
by various growth regulators. This was visually observed by
noticing a higher proportion of alfalfa somatic embryos showing bipolarity and increased cotyledon development. One way
to quantify the relative development of somatic embryos is to
measure their subsequent conversion to plantlets with roots
and true leaves (16). Results of representative conversion
experiments are shown in Table V for various auxin treatments. In all cases, there is an increase in the rate of embryo
conversion to plants for 4-(2,4-dichlorophenoxy)butanoic
acid and the branched side chain analogs of the phenoxy
acids. All of these improvements are statistically significant
except for (+)-2-(2,4-dichlorophenoxy)propanoic acid. This
can be interpreted to mean that the embryos that regenerate
from these cultures are better developed than those from 2,4D-treated cultures.
Somatic Embryo
'M
Experiment 1
2,4-D
Rac 2-(2,4,5-dichlorophenoxy)
propanoic acid
Experiment 2
2,4-D
50
10
50
100
500
262 ± 19
184 ± 10
173 ± 10
224 ± 14
50
3
129 ± 30
71 ±18
139 ±± 20
51 9
88 ± 11
10
Rac 2-(2,4,5-trichlorophenoxy)
propanoic acid
100
300
number
212±11
Celery and Lettuce Cell Cultures
Experiment 3
50
2,4-D
1
4-Methyl-2-chlorophenoxyacetic acid
3
10
50
100
300
Rac 2-(4-methyl-4-chlorophenoxy)propanoic acid
3
10
50
100
300
1
232 ± 15
0
0
12 ± 5
190 ± 17
136 ± 28
170 ± 13
0
0
8±3
83 ± 14
179 ± 20
137 ± 11
Several of the growth regulators that were found to be
effective in alfalfa were tested in celery and lettuce cultures
(Table VI). With celery, a standard subculture and induction
treatment described by Orton ( 14) gave only one embryo per
plate in a replicated experiment, whereas 4-(2,4-dichlorophenoxy)butanoic acid, racemic 2-(2,4-dichlorophenoxy)propanoic acid, (+)-2-(2,4-dichlorophenoxy)propanoic acid, and
racemic 2-(2-methyl-4-chlorophenoxy)propanoic acid all gave
significantly higher embryogenesis. In lettuce, a standard lettuce regeneration procedure for somatic bud formation using
3 IAM 2-chlorophenoxyacetic acid [V] yielded no somatic
embryos, whereas treatments using racemic 2-(2,4-dichlorophenoxy)propanoic acid all yielded somatic embryos. These
results demonstrate in two additional species unrelated to
alfalfa that the same phenoxy acid growth regulators have a
Table ll. The Effect of 2,4-D [I], Racemic 2-(2,4-Dichlorophenoxy)propanoic Acid [V], Racemic [XI], (+) [XII], and (-)-2-(2,4,5Trichlorophenoxy)propanoic Acid [XIII] on the Induction of Alfalfa Somatic Embryos
Phenoxy Acid
Somatic
Concentration
Embryo
Number
AM
2,4-D
Rac 2(2,4-dichlorophenoxy)propanoic acid
233 ± 14
177 ± 86
50
100
Enantiomers
2-(2,4,5-Trichlorophenoxy)propanoic acid
0.5
1.0
10
50
100
300
(+)
64 ± 7
83 ± 8
140 ± 67
140±6
170 ± 17
135 ± 15
Racemic
(-)
28 ± 5
63 ± 7
160 ± 10
188±16
175 ± 14
94 ± 12
0
0
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1 ±0
12±7
3±3
0
116
STUART AND McCALL
Plant Physiol. Vol. 99, 1992
Table IV. Summary of the Induction of Alfalfa Somatic Embryogenesis by Various Synthetic Growth Regulators
Summary includes data from this report and ref. 9 based on the bioassay described in "Materials and Methods."
Growth Regulator
Form
Biological Activity
Optimum
JM
AM
2,4-D
4-Chlorophenoxyacetic acid
Picloram
2,4,5-Trichlorophenoxyacetic acid
+
+
+
+
25-75
10-500
75-500
25
10-100+
75-200
200-500
10-100+
3(2,4-Dichlorophenoxy)propanoic acid
4(2,4-Dichlorophenoxy)butanoic acid
5(2,4-Dichlorophenoxy)pentanoic acid
2(2,4-Dichlorophenoxy)propanoic acid
+
100-500
50-100
+
+
Not available
+
+
50-100
50-100
10-500
10-500
+
+
10-100
10-300
50
100-300
100-300
0.5-300+
0.5-300+
10-100
50-500
50-500
+
+
+
50-100
50-100
50-100
10-300+
10-300+
10-300+
Racemic
+
2(2,4,5-Trichlorophenoxy)propanoic acid
Racemic
+
+/-
2(4-Chlorophenoxy)propanoic acid
Racemic
+
2(4-Methyl-2-chlorophenoxy)propanoic acid
2(4-Methyl-2-chlorophenoxy)propanoic acid
Racemic
Racemic
+
positive effect
with 2,4-D.
on
somatic embryo development compared
DISCUSSION
Exhaustive studies on the structure and activity of phenoxy
acid growth regulators have been reported for oat coleoptile
elongation, wheat root growth inhibition, and the flax root
test (20-22). These studies showed that phenoxy acids with
even numbers of carbons between the phenoxy ring and the
carboxyl group are inactive (e.g. 3-(2,4-dichlorophenTable V. Effect of Selected Phenoxy Acid Growth Regulators on the
Conversion of Alfalfa Somatic Embryos to Plantlets
Concentration Conversion
%oEmbrso
Phenoxy Acid
M
Experiment 1
2,4-D
4(2,4-Dichlorophenoxy)butanoic
acid
Experiment 2
2,4-D
Rac 2(2,4-Dichlorophenoxy)propanoic acid
Experiment 3
2,4-D
Rac 2(2,4-dichlorophenoxy)propanoic acid
50
50
100
50
10
50
oxy)propanoic acid), 5-(2,4-dichlorophenoxy)pentanoic acid,
etc.) for cell elongation, whereas those with an odd number
of carbon atoms (e.g. 2,4-D, 4-(2,4-dichlorophenoxy)butanoic
acid, etc.) are active for cell elongation. These studies also
showed that if the active compounds are further substituted
with a methyl or an ethyl group at the carbon adjacent to the
phenoxy ring, these structures are optically active and the
racemic and (+) enantiomers are active in growth tests but
the (-) forms are inactive.
The present study represents the first report in which the
structure-activity relationships of similar phenoxy acid growth
regulators have been studied for in vitro somatic embryogenesis, a system of plant development. These results are similar
Table VI. Effect of Various Phenoxy Acid Growth Regulators on
Celery and Lettuce Somatic Embryogenesis
25 ± 4
40 ± 4
48 ± 5
22 ± 4
45 ± 7
49 ± 8
50
100
23 ± 6
41 ± 4
(+)2(2,4-dichlorophenoxy)pro-
100
33 ± 5
Rac 2(2,4,5-trichlorophenoxy)propanoic acid
Rac 244-methyl-2-chlorophenoxy)propanoic acid
100
41 ± 4
100
44 ± 5
panoic acid
Activity Range
Phenoxy Acid
Concentration Embryos
Plate per
M
Celery
2,4-D
4-2,4-Dichlorophenoxy)butanoic acid
Rac 2(2,4-dichlorophenoxy)propanoic acid
(+)2(2,4-Dichlorophenoxy)pro-
panoic acid
Rac 2(4-methyl-2-chlorophenoxy)propanoic acid
Lettuce
2-Chlorophenoxyacetic acid
Rac 2(2,4-dichlorophenoxy)propanoic acid
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Copyright © 1992 American Society of Plant Biologists. All rights reserved.
2.25
1
2.25
1
2.25
1
2.25
1
2.25
3
3
1
109
51
93
101
89
53
134
43
0
8
SOMATIC EMBRYOGENESIS WITH ANALOGS OF 2,4-D
in two important respects to the cell elongation studies. First,
in alfalfa cell cultures (Table I), the phenoxy acids with an
odd number of carbon atoms between the phenoxy ring and
the carboxyl group (e.g. 2,4-D, 4-(2,4-dichlorophenoxy)butanoic acid) are also active for the induction of cell
differentiation, whereas those with even numbers of carbons
(e.g. 3-(2,4-dichlorophenoxy)propanoic acid), 5-(2,4-dichlorophenoxy)pentanoic acid) are completely inactive. Second,
we find that for optically active branched side chain structures
(e.g. 2-(2,4,5-trichlorophenoxy)propanoic acid)), the racemic
and the (+) enantiomer are active for inducing tissue differentiation but the (-) enantiomers are essentially inactive
(Tables III and IV). We do find very limited activity for
induction of somatic embryogenesis in some experiments for
the (-) form (Table III) but attribute this response to a slight
contamination of the (-) form with the (+) form, which
sometimes occurs during recrystallization of the stereoisomers
from racemic mixtures. These results suggest that the same
basic mechanism of auxin perception may be active in inducing cell elongation as well as cellular differentiation.
Two surprising results were observed, however, which could
not be predicted from the earlier results with cell elongation.
First, it is interesting to note that 4-(2,4-dichlorophenoxy)butanoic acid is active in alfalfa cell cultures (Tables I,
IV, and V) but shows no herbicidal activity toward alfalfa in
the field (23). The selective herbicidal activity of 2-(2,4dichlorophenoxy)butanoic acid in the field against broad leaf
competitors is thought to be due to a lack, in alfalfa, of the ,Boxidation pathway that would convert 4-(2,4-dichlorophenoxy)butanoic acid to 2,4-D, the herbicidally active form (26).
Based on this selective herbicidal activity, we originally expected 4-(2,4-dichlorophenoxy)butanoic acid to be inactive
for the induction of somatic embryogenesis in alfalfa cell
cultures. The results we observe, however, suggest that either
that (a) the ,B-oxidation pathway is present in alfalfa cell
cultures but not in the mature plant so that 4-(2,4-dichlorophenoxy)butanoic acid is broken down to the inductive 2,4D, or that (b) 4-(2,4-dichlorophenoxy)butanoic acid itself is
an active inducer of somatic embryogenesis.
Second, we find that chain extension (e.g. 4-(2,4-dichlorophenoxy)butanoic acid) or side chain branching (e.g. 2-(2,4dichlorophenoxy)propanoic acid, 2-(2,4,5-trichlorophenoxy)propanoic acid, etc.) result in improved embryo development compared with induction with 2,4-D (Tables V and
VI). In alfalfa, this effect was not reflected in improved
somatic embryo yields upon regeneration (Tables I-IV) but
is consistently observed when conversion (germination) to
plantlets is measured (Table V). In celery and lettuce, use of
these compounds improve the overall yield of somatic embryos (Table VI). In practical terms, it is not necessary to use
the (+) active form of the branched side chain phenoxy acids
to observe improved embryo development; racemic mixtures
will work just as well. This result supports our conclusion that
(-) enantiomers are completely inactive because racemic
mixtures neither reduce the yield of embryos nor inhibit the
later embryo development compared with the purified (+)
enantiomer. Growth studies using the pea stem curvature and
the Avena coleoptile test by Smith (17, 18) suggest that the
(-) enantiomers will antagonize the action of the (+) enan-
117
tiomer. We find no evidence for such an antagonism in
developing cell cultures.
Finally, one can speculate why side chain branching and
chain extension result in improved yields of somatic embryos
and subsequent improvements in embryo development. We
suggest that acceleration of the metabolism or breakdown of
these growth regulators may be responsible for this effect.
Evidence for this hypothesis comes from two types of studies.
First, it is well known that 2,4-D prevents the early stage
development of somatic embryos if added to the regeneration
medium (3) and yet it is widely used for induction of embryogenesis (6, 8, 9). Induction of alfalfa cell cultures with
suboptimal concentrations of 2,4-D, however, causes lower
yields of early stage embryos but also increases the developmental maturity of the resulting embryos as measured by
increased storage protein deposition and conversion (9). This
suggests that if too much 2,4-D is carried over from the
induction medium, either in an intracellular or an extracellular pool, the resulting somatic embryo will display arrested
or abnormal development. Second, studies on the rates of
metabolism of 4-(2,4-dichlorophenoxy)butanoic acid (24) and
2-(2,4-dichlorophenoxy)propanoic acid (25) have been compared to that of 2,4-D. These studies find that the former
molecules are metabolized more quickly than 2,4-D. If increased metabolism of the phenoxy growth regulators is responsible for the improved somatic embryo development seen
in this report, this suggests that the appropriate design of the
inductive molecule itself could result in strong induction of
early stage somatic embryos followed by rapid metabolism of
the phenoxy acid growth regulator to an inactive form that
would promote better somatic embryo maturation and development. The demonstration of the metabolic fate of these
phenoxy growth regulators in cell cultures awaits further
investigation.
ACKNOWLEDGMENTS
We appreciate the gifts of (+)-2-(2,4-dichlorophenoxy)propanoic
acid from Prof. K.V. Thimann as well as (+)- and (-)-2-(4-chlorophenoxy)propanoic acid from Dr. V. Tortorella. The efforts of Steve
Strickland are recognized and appreciated for his contribution to the
culture of lettuce embryos. Also appreciated were the discussions with
Ralph Mumma of Pennsylvania State University on the metabolism
of phenoxy acid growth regulators.
1.
2.
3.
4.
5.
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118
STUART AND McCALL
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