BIOLOGIA PLANTAR~I"M (PRAHA)

11 (3) : 2 0 8 - - 2 1 5 , 1969

Role of Growth Regulators in Differentiation Processes

of Maize (Zea Mays L.) Organs

Z. S L A D K ~
Laboratory of P l a n t Physiology and Anatomy, Faculty of Sciences,
J. E. Purkyn~ University, Brno*
Received April 1, 1968

Abstract. The following paper deals with the character of endogenous auxins and gibberollin-
like substances in the maize tassel and ear primordia during differentiation. Using bioassay the
character of substances extracted from tassel prlmordia, internodos below the tassel, ear pri-
mordia and stem base was determined and correlated with the course of morphogenesis and
differentiation. A low level of auxins and a high content ofgibberellin-liko substances accompanies
the differentiation of terminal tassel. The differentiation of an ear is associated with an increment
in auxin content while the level of gibberellin-liko substances decreases. The character of grow th
substances in primordia remains practically unchanged in the course of further differentiation.
The inhibitions appear in the plant and probably start numerous morphological reductions in the
pistillate inflorescence structure or inhibit the growth of lateral primordia on the stem etc. The
t r e a t m e n t of plants with maleic hydrazide at the beginning of tassel differentiation shifts the
normal levels of endogenous regulators and brings about the transformation of tassel primordia
into an ear. This transformation is accompanied by a marked rise in gibberollin-liko substances,
by an increment in auxins and the appearance of inhibitors.

The coordination of all the processes and the harmonic step by step develop-
ment in the morphogenesis of plant organs is quite a puzzling phenomenon.
We do not understand the mechanisms governing cell division and ontogene-
tical development at the moment. The problem lies evidently in the difficulty
of characterizing the ]evels of organization where the processes are taking place
(ZuBAY 1964). Morphogenesis may be considered as a result of many bioche-
mical processes anchored in the genetical backround of the plant. HESLOP-
HAR~SO~ (1964) points out that the level of growth regulators in the mer-
istem before differentiation influences the pattern of further development.
In our previous work (SLADK~ 1966) we paid attention to the role played
by substances of auxin character in the differentiation of tassel and ear pri-
mordia. The aim of our present work was to study the link between endo-
genous auxins and gibberellins and some morphogenetical and differentiation
processes in maize. We were also interested in the role played by these sub-
stances in the induction of abnormalities.
Address: Kotlg~skg~ 2, Brno, Czechoslovakia.

208
 GROWTH R E G U L A T O R S I N MAIZE 209

Material and Methods

The single hybrid of maize (WIt • W9) was used in the experiments. In
the years 1965 to 1967 plants were grown in the field from the beginning of
May to the end of September. The auxins and gibberellin-like substances
were determined in the tassel primordia(1), in the internodes below the tass-
el (2), in ear primordia (3) and in the internodes at the stem base (4). The
first series of experiments was carried out with plants 35 4- 3 days old with
terminal tassel primordia 1.5 cm. long a n d t h e phase of the beginning of stam-
inate floret differentiation. Plants 49 ~ 3 days old were used for the second
series of experiments. In these plants the ear primordia in the middle of the
stem were 1.5 cm. long and t h e y reached the phase of the beginning of pistil-
late floret differentiation. The changes in growth substance content after
0-1 per cent maleie hydrazide t r e a t m e n t were determined in plants 49 4-3 days
old. When repeating the experiments special attention was paid to use the
primordia not only of the same age b u t also of the same developmental phase.
Peroxide-free ether was used for extraction according to BENTLEY and
HOUSLEY (1954) and KEFFO~D (1955). The original method was modified in
some details. 10 g fresh weight of excised primordia were homogenized and
extracted three times with 100 ml cooled ether. The combined extracts were
concentrated and the residue dissolved in 1.5 ml ethanol. 0.5 ml was applied
to W h a t m a n n No. 1 paper and a mixture of isopropanol-ammonia-water
(8 : 1 : 1) used for chromatography. 1.0 and 0.1 p p m solutions of IAA were
used as a standard. Chromatograms were divided into ten zones and eluted
using 3 per cent sucrose. Oat (Arena sativa L.) cv. ~esk~ ~lut~ was em-
ployed for coleoptile segment bioassay. The segment's elongation was ex-
pressed in histograms as percentage of control elongation. Each histogram
represents the mean value of at least three experiments with 3.3 g primordia
fresh weight.
Gibberellin-like substances were determined in 2 g fresh weight of appro-
priate organs using TLC technique according to SEmaD~.B, GBoss and
SCHBv.mER (1962). The volume of extracts was reduced b y evaporation of
methanol the residue dissolved in 0.5 ml ethylacetate and 0.3 ml applied on
silicagel G (Merck) thin layer. Gibberellie acid of Polish origin (Gibreskol)
in concentration 0-1 and 1-0 p p m was used as a standard. Chromatograms
were developed in solvent system chloroform-ethylacetate-ice acetic acid
(60 : 40 : 5) and the gibberellin-like activity of different zones examined b y
lettuce hypocotyl bioassay (cv. Stupick~ Kamens The results of the ana-
lysis are expressed in histograms as a percentage of control elongation. E a c h
histogram represents the mean value of at least three experiments with 2 g
fresh weight.
Plants were sprayed with 0.1 per cent maleie hydrazide prior to differen-
tiation of staminate spikelets in the tassel primordia. The t r e a t m e n t was
repeated after six days. The course of morphogenesis and differentiation was
followed simultaneously with the changes in growth regulators level. The
photograph represents differentiation of an ear and the numbers indicate
regions of sampling for endogenous regulator extraction.
 210 z. SLADKY

Results

The character of auxins and gibberellin-like substances at the beginning

of spikelet and floret differentiation in a tassel is represented in Fig. 1. The
upper histograms present data on auxin bioassay, the lower ones on gibberel-
lin bioassay.
The beginning of terminal tassel differentiation is associated with low auxin
level (1). In the internodes below the tassel (2) there are regions of stimulative

Fig. 1. Character of endogenous regulators in maize primordia prior to differentiation of stami-

nate florets of the tassel. The histograms on the left side above represent auxins, t h e
histograms below gibberellin-liko substances in tassel primordia (1), in internodes below
tassel (2), in ear primordia in the middle p a r t of the stem (3) and a t the stem base (4).
The effect of 1.0 and 0.1 ppm of appropriate standard is indicated on the ordinate.
 G R O W T H R E G U L A T O R S I N MAIZE 211

as well as inhibitive character especially at R f 0.2, 0.9 and 1.0. Ear primordia
(3) contain substances of stimulative nature. R f 0-5 and 0.6 correspond to IAA
in a concentration higher than 1 mg/1. There is only low auxin activity at the
stem base (4).
The lower histograms represent results on gibberellin-like substance esti-
mation. As shown in histogram 1, the highest level of gibberellin was found in
the tassel primordia. The activity at R f 0.3 and 0-4 corresponds to the effect
of approximately 0.1 mg/1 GAs. There are zones of stimulative character even
at the start and in the front of a chromatogram. The internodes below the
tassel (2) contain no substances causing lettuce hypocotyl elongation. The ear
primordia (3) contain less substances of stimulative character than tassel
pr]mordia. Substances from stem base (4) are exclusively of inhibitive char-
acter.
Fig. 2 shows the result of analyses done 14 days later, at the time of spikelet
and f[oret differentiation in the ears. There exists a certain correspondence
with the previous results, the inhibitions being more pronounced.
The level of auxins in the tassel primordia (1) remains practically unchan-
ged. In the internodes below the tassel (2) quite a marked inhibition appears
at R f 0.2, 0.4 and 0.5. The auxins in ear primordia (3) even increased. The
auxin effect is quite clear-cut at R f 0.5 and 0.6 where the stimulation corre-
sponds to an IAA concentration of 6.5 mg IAA/10 g fresh weight of ear pri-
mordia. Slight inhibition at R f 0.2 appeared. The situation at the stem
base (4) remains practically unchanged.
The lower histograms again represent gibberellin-like substances. In the
tassel (1) the stimulation at R f 0.1 and 0-4 is accompanied b y pronounced
inhibition at R f 0-6. There is a rise in inhibitions even in the internodes below
the tassel (2), at R f 0-1 and especially at 0.5 to 0-7. I n ear primordia (3) the
gibberellin-like substances tend to decrease and zones with inhibitive char-
acter appear. At the stem base (4) the inhibitions are less marked in compa-
rison with the previous situation, neverthless one half of R f zones re~ eal an
inhibitive character.
The changes in endogenous regulator level caused b y two maleie hydrazide
treatments prior to terminal tassel differentiation are shown in Fig. 3. It fol-
lows from histograms, t h a t the treatment brought about m a n y fundamental
changes. Inhibitions appear in the tassel primordia and a slight increment in
auxins m a y be observed. There is a drop in inhibitions in the internodes below
the tassel (2). The content of substances with stimulative character in ear
primordia (3) decreases and new inhibitive regions appear. At the stem base (4)
zones of stimulative and inhibitive character interchange along the whole
chromatogram. The comparison with Fig. 2 shows clearly the destructive
effect of maleic hydrazide on the normal level of endogenous substances with
auxin character.
In the lower histogram the effect of maleic hydrazide on the level of
gibberellin-like substances is shown. The effect is even more pronounced than
in the previous case and a general increment in substances of stimulative
character m a y be observed. The highest values were found for tassel primordia
where zone 1 surpasses the activity of 1 mg/1 GAs. R f 0.3 and 0-4 correspond
approximately to the effect of 0.1 mg/1 GAs. There is an increased stimulative
activity even in the internodes below the tassel (2) and in ear primordia (3}
212 z. SLADKY

Fig. 2. C h a r a c t e r of e n d o g e n o u s r e g u l a t o r s in m a i z e p r i m o r d i a 14 d a y s a f t e r first d e t e r m i n a -
t i o n i.e. a t t h e t i m e of e a r pistillate florets differentiation. T h e d a t a are p r e s e n t e d in t h e
s a m e w a y as in Fig. I. T h e p h a s e of d e v e l o p m e n t is c h a r a c t e r i z e d b y a p h o t o g r a p h
o f a m a i z e p l a n t w i t h d i f f e r e n t i a t e d t a s s e l a n d pistillate florets of t h e e a r in t h e c o u r s e
o f differentiation.

where already some inhibitions may be found. There was no inhibition in the
samples from the stem base as in the previous cases by control plants.
From the morphological point of view maleic hydrazide brought about the
 GROWTH R E G U L A T O R S LN MAIZE 213

Fig. 3. Treatment with 0-1 per cent maleic hydrazido causes changes in the level of endogenous
auxins (above) and gibberellin-like substances (below) in differentiating tassel pri-
mordia (1), in internodes below the tassel (2), in ears in the middle part of the stem (3)
and at the base of the stem (4). The photograph shows transformation of the tassel
in pistillate ear.

feminization of tassel florets and even the tassel primordia were transformed
into a typical ear (Fig. 2). The appropriate treatment with maleic hydrazide
elongates internodes below the tassel and causes uniform growth of several
ears on the stem.
214 z. SLADK~,"

Discussion

The results indicate t h a t a high degree of specialization in the structure of

maize organs is based on a perfectly developed system, with auxins, gibber-
ellin-like substances and inhibitors playing an important role. The method
of bioassay used for evaluation of chromatographic zones enables us to char-
acterize endogenous substances and yields data for elucidation of the pattern
of different primordia differentiation. No definite conclusions m a y be drawn
in respect to possible interaction between substances with stimulative and
inhibitive character and of free and bound forms of growth substances as
conceived by LIBBERT (1954).
The results indicate t h a t the level of growth regulators is rather constant
during the whole course cf differentiation. We found t h a t a differentiation of
pistillate inflorescence is accompanied by a higher auxin level which is in
accordance with our previous d a t a ( S L A D K Y ~ 1966) and those of CONRAD and
~Y[OTHES (1961), who have found 30 times more auxins in pistillate t h a n in
staminate hemp plants.
There is a rise in inhibitions during maize primordia differentiation. Nothing
definitive can be said about the character of these inhibitions. The difficulty
lies, in the first place, in a lack of adequate methods and no standards for
comparison are available. I t seems t h a t the inhibitors play a role at least of
similar importance to t h a t of stimulators. According to I-I~SLOP-I-IARRmO~r
(1964) the feminization of maize tassel florets by short d a y is accompanied
rather by inhibitor accumulations, t h a n by increment of native auxins level.
GALUN (1959), on the other hand, ascribes a limiting role to auxins in the
differentiation of cucumber inflorescence primordia. I-[e came to the conclu-
sion t h a t an addition of IAA to the cultivating medium aids the carpel devel-
opment and inhibits the growth of stamens. In intact cucumber plants gibber-
ellin acts in a way opposite to auxins and inhibits the development of pistil-
late flowers by the simultaneous enhancement of staminate primordia growth.
I:)ILET'S (1961) view on the synergic effect of gibberellic acid and auxin,
which is often cited in the literature, is probably more valid for growth t h a n
for differentiation. The relation between auxin and gibberellin seems to be
rather antagonistic in our experiments. Low auxin level and high gibberellin
content is associated with tassel differentiation and the reverse is true for
ear differentiation.
The appropriate t r e a t m e n t with maleic hydrazide brings about alteration
in the level of endogenous regulators and changes the morphological struc-
ture. It seem t h a t the shifts in growth regulator levels are related to morpho-
logical changes in a way similar to t h a t in previous eases. This kind of data
m a y be considered as a further indication of a link between endogenous
regulators and differentiation processes. In accordance with TELTSCHEROVJ~'S
findings (1968) we m a y say t h a t the determination of growth substance level
relations is a more important indicator of differentiation t h a n the determi-
uation of individual substance concentration.
 GROWTH REGULATORS IN MAIZE 215

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H a n f p f l a n z e n . -- N a t u r w i s s e n s c h a f t e n 48 : 2 6 - - 27, 1961.
GALUN, E.: E f f e c t s of gibberellic acid a n d n a p h t h a l e n e acetic acid o n s e x e x p r e s s i o n a n d s o m e
m o r p h o l o g i c a l c h a r a c t e r s in t h e c u c u m b e r p l a n t . -- P h y t o n 13 : l - - 8 , 1959.
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n a t u r e l s de la croissance v6g6tale. -- Colloques i n t e r n . P a r i s pp. 6 4 9 - - 6 6 4 , 1964.
KE~FORD, •. P.: T h e g r o w t h s u b s t a n c e s s e p a r e d f r o m p l a n t e x t r a c t s b y c h r o m a t o g r a p h y . ][ f- I I .
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p e n h e m m u n g . -- P l a n t a 44 : 2 8 6 - - 3 1 8 , 1954.
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SLADK'2, Z.: T h e effect of g r o w t h s u b s t a n c e s on t h e d i f f e r e n t i a t i o n of floral s h o o t s of maize. --
A c t a U n i v . Carol. Biol. ( P r a h a ) , Supl. 1966 1/2 pp. 1 3 1 - - 1 3 3 , 1966.
TELTSCHEROV.~_, L.: C h a n g e s in t h e level of e n d o g e n o u s gibberellins a n d a u x i n s in a p m a l b u d s
of Chenopodium rubrum L. a f t e r a p p h c a t i o n of g r o w t h s u b s t a n c e s r e v e r s i n g t h e effect o f
( 2 - c h l o r e t h y l - t r i m o t h y l a m m o n i u m chloride (CCC) on flowering. - Biol. P l a n t . 10 : 305
310, 1968.
ZUBAY, G.: T h e m o l e c u l a r basis of differentiation. -- B r o o k h a v e n S y m p o s i a in Biol. 1 6 : 1 7 0
to 178, 1964.
Z. SLAnKS~, L a b o r a t o ~ fyzmlogio a a n a t o m m r o s t l i n P H r o d o v S d e e k 6 f a k u l t y , B r n o : ~loha rfsto-
v~eh r e f u h l t o r h v proeeseeh difereneiaee or.qfin6 k u k u H e e Z e a m a y s L . -- Biol. Plant. 11 : 208
a~ 215, 1969.
P~edlo~enA prAce sleduje c h a r a k t e r e n d o g e n n i c h 1Atek a u x i n o v 6 a gibcrelov6 p o v a h y v zAkla-
d e c h orghnfl kuku~ice b S h e m diferenciace praw l a t y a p e s t i k o v 6 pal!co. B i o l o g i c k ~ m testo-
vg~nim b y l zji~fov~n c h a r a k t e r o x t r a h o v a n : @ h ls ze z~kladfl l a t y , intornodii p o d latou, z h k l a d 6
palie a b a z e st6bla a jo u v h d ~ n do v z t a h u s p r f l b ~ h e m morfogonezo a diforonciace. N i z k h h l a d i n a
a u x i n f l a vysok:~ o b s a h 1htek giberelov6 p o v a h y p r o v h z l diferenciaci t e r m i n h l n [ praw l~ty.
Diferenciace p e s t i k o v 6 pal!co je c h a r a k t e r i s o v g m a z v : ~ c n i m o b s a h u l~tek a u x i n o v 6 p o v a h y a sni-
~ e n i m h l a d i n y giberelinfl. V prflb6hu dal~i diferenciace sc c h a r a k t e r 1Atek v z~kladech p o d s t a t n 8
n e m ~ n i . DochAzi k narflstAni inhibic v rostlinS, kter6 p r a v d 6 p o d o b n 6 z a v h d i ~adu m o r f o l o g i e k ~ c h
r e d u k c i ve s t a v b 6 p e s t i k o v 6 h o k v ~ t e n s t v i , n e b o i n h i b u j i rflst laterAlnich z~kladfl n a st6ble a p e d .
Post~ik rostlin m a l e i n h y d r a z i d e m n a p o 6 s k v 6 t n f diferenciace l a t y poru~f pf-irozenou h l a d i n u
regul~torfl v rostlin6 a vyvolA p i ' e m 6 n u zhkladfl pra~nikov6 l a t y v p o s t i k o v o u palicL M o t a m o r f o z a
je p r o v h z e n a p o d s t a t n ~ m z v : ~ e n i m 1Atek giborelov6 p o v a h y , s t o u p n u t i m h l a d i n y auxinf~ a v)~-
s k y t e m 1Atek inhibi6ni p o v a h y .
3. CJIh~[~tI, dIaGopaT()|)|t~l ~H3tlOJ[Ol'Ill[ H ~ilIdTOMIII~I pa('Telllti;l, (pat~y~bTeT OCTO('TBO31I~IHHH,
Epno: POJIb p e r y ; m T o p o B pocTa B n p o [ t e c c a x ~ii~)~)epeliltnlalDl/! o p r a n o u nyuypyab~ Zea
maysL. Biol P l a n t . ! 1 : 2 0 8 - 2 t 5 , 1969
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