BIOLOGIA PLANTARUM (PRAttA)

1 (1) : 16--21, 1959

Observation o[ IEPM Values o~ Growing Points ot Flower Buds

o~ the Apple during the Period
before their Morphological Differentiation

BO~IVOJ HO~AVKA
Department of Plant Physiology, Institute of Biology, Czechoslovak Academy of Sciences,
Praha

Received January 30, 1958

Souhrn

Byly zjih~ov~ny hodnoty IEP M u meristomatick:~ch pletiv vzrostn:~eh vrehoh2

kvStnich pupenfl, u flo6mu a xyl6mu letorostfl a brachyblastfl u t~i odrfld jabloni:
Prflsvitn6 iflut6, Lord Lambourne a Watervlietsk6.
l~ezy 50 p silnS, zhotoven6 ru6nim mikrotomem, byly 10 rain. fixovs v 70%
ethanolu a pot~ umistiiny na 2 hod. do ~ady pufrovan~ch (citrAtov6 pufry podle
S6RENSE~rA) roztokfi kysel6ho fuchsinu a anilinov6 zeleni (konc. 1 : 10 000), stup-
fiovan)~ch po 0,2 pH. DAle byly ~ezy p~emist~ny na pill hodiny do bezbarv~ch
pufrfl stein6 hodnoty pH.
Za isoelektrickou oblast bylo poklhdAno zbarveni pletiv od 6ervcnofialov~ho do
modrozelen~ho odstinu. St~ednl hodnota isoelektrick8 oblasti byla zna6ena jako
IEP M.
PrflbSh hodnot I E P M vzrostn~ch vrcholfl pupenfl se vyzna6oval dv6ma fhzeml.
V prvnl fAzi od po6Atku studia do 9. 7. 1957 zfistAvaly hodnoty I EP M na firovni
3,0 s maxims odchylkami 0,1. Ve druh6 fs od 9.7. do 6.8. 1958 doyle k posunu
hodnot IEP M sm~rem k vyh~imu pH. Posun v~ak byl doSasn6ho rs Po dosa~ienl
hranice u odrfldy Prdswtn6 ~lut6 3,5, Lord Lambourne 3,6 a Watervlietsk~ 3,3
doyle k posunu hodnot I E P M na pflvodni firovefi (3,0). V obdobl zp~tn6ho posunu
hodnot IEP M k oblasti o ni~im pit byl zji.~t6n po6Atek morfologick6 difereneiace
vzrostn:~ch vrcholfl.
U flo6mu a xyl~mu brachyblastfi a letorostfl mime podobn:~ posun hodnot I EP M
k oblasti o vyhw pI-I v obdobi diferenciace vzrostn:~ch vrcholfl byl zji~t6n posun
jew v obdobi zastaveni dlou~iv~ho rflstu na za6s l~ta.

Summary
1. D u r i n g t h e p e r i o d f r o m J u n e 11 t o A u g u s t 6, 1957 I E P M v a l u e s w e r e
recorded for the meristematic tissues of growing points of apex buds of brachy-
blasts a n d for p h l o e m and x y l e m o f brachyblasts and growing spurs of apple
varieties Transparent Yellow, Lord Lambourne and Watervliet.
2. F o r a p e r i o d o f 7 - - 1 4 d a y s b e f o r e t h e m o r p h o l o g i c a l d i f f e r e n t i a t i o n
of growing points of flower buds the shift of IEP~ values towards the area
of higher p H was observed. This shift was f o u n d b o t h in t h e m e r i s t e m a t i c
tissues o f growing points and in the p h l o e m and x y l e m of b r a c h y b l a s t s and
growing spurs.

16
 IEP M VALUES OF GROWING POINTS OF FLOWER BUDS 17

3. After reaching a certain limit a shift of IEPM values back to the area
of lower p i t at the original level was observed.

Introduetion

The development of flower buds of the apple has already been adequately
studied from the morphological aspect. The period of the start of morpho-
logical differentiation of flower buds has already been established for many
"varieties (Ho~AVKA 1957) and individual phases of the formation of flower
organs have been recorded. Physiological processes taking place in the growing
points during the period before their differentiation have, however, been stud-
ied only to a limited extent. Since these processes include among others
physico-chemical processes, the author turned his attention to following
the values of the isoelectric area of growing points of buds and, in addition,
recorded the values for the phloem and xylem of braehyblasts and growing
spurs of apple trees. In the course of the investigation, however, the extent of
the isoelectric area came to involve a varied number of tenths of p H and,
therefore, the median value, commonly ,known in the literature as index
IEPM, was employed to facilitate a comparison of results.
The values of the isoelectric point (IEP) have been mainly determined
for micro-organisms and b y many authors, from the methodical aspect, for
the epidermis of onion (Allium cepa) leaves. Some authors have studied the
values of I E P in certain plants during growth and development (KONAREV
1948a, b), others have investigated the course of I E P values in different
parts of the cell ( S T R U G G E R 1932, ])RAWERT 1937 and others). Their results
are, however, markedly divergent. From the available literature we know of
only one investigation of values of I E P for apple buds ( T S E L N I K E R 1949).

Materials and Methods

I E P M v a l u e s , as h a s b e e n a h ' e a d y m e n t i o n e d , were d e t e r m i n e d for t h e m e r i s t e m a t i e t i s s u e s
of t h e g r o w i n g p o i n t s of t h e a p e x b u d s of b r a c h y b l a s t s a n d for p h l o e m a n d x y l e m of b r a c h y -
b l a s t s a n d g r o w i n g s p u r s in t h r e e varieties of apple ( T r a n s p a r e n t Yellow, L o r d L a m b o u r n e a n d
W a t e r v l i e t ) . Sections 50 p thick, c u t b y a h a n d m i c r o t o m e , were fixed w i t h 7 0 % e t h a n o l , w h i c h
according to ZEIaER (1930) is t h e m o s t suitable fixative since its c a u s e s t h e least s h i f t o f I E P
o f t h e s p e c i m e n s s t u d i e d f r o m t h e a c t u a l values. A c c o r d i n g to SCHWANTES (1952) o t h e r fixatives,
s u c h as m e t h a n o l , s u b l i m a t e , osmic acid, C a r n o y m i x t u r e , s h i f t I E P t o w a r d s h i g h e r p H v a l u e s
as c o m p a r e d w i t h 70~o e t h a n o l while p o t a s s i u m d i c h r o m a t e , f o r m a l d e h y d e a n d f o r m a l d c h y d -
e t h a n o l shift it t o w a r d s lower p H v a l u e s .
T h e fixed sections were s t a i n e d in a buffered solution of acid f u c h s i n c a n d aniline green.
C i t r a t e buffer after SORENSEN, w i t h 0"2 p H i n t e r v a l s w a s u s e d to stabilize t h e m i x t u r e . T h e
a c t u a l p H v a l u e s of t h e buffers were d e t e r m i n e d p o t e n t i o m e t r i c a l l y a n d d i d n o t differ b y m o r e
t h a n -J:0.03 p H f r o m t h e t a b u l a r values. E a c h of t h e s t a i n s u s e d w a s p r e s e n t in t h e b u f f e r in
c o n c e n t r a t i o n of 1 : i0 000. H i g h e r c o n c e n t r a t i o n s , as s h o w n b y m o d e l e x p e r i m e n t s , shift t h e
I E P M v a l u e s towaz'ds t h e a r e a of lower p H .
A f t e r fixation, t h e sections were s t a i n e d for 2 h o u r s in t h e g i v e n s t a i n i n g m i x t u r e a n d t h e n
rinsed for 30 m i n u t e s in colourless buffer s o l u t i o n s of t h e s a m e p H value. T h e isoelectric a r e a
w a s i n d i c a t e d b y s t a i n i n g of t h e t i s s u e s f r o m reddish-violet to bluish-green. As a r e s u l t of t h e i r
positive charge t h e t i s s u e s were s t a i n e d r e d o n l y b y acid f u c h s i n e a t lower p H t h a n t h e lower l i m i t
of t h e isolelectric area, while at h i g h e r p H t h a n t h e u p p e r limit of t h e iso~lectric a r e a t h e y were,
18 B. HO~AVKA

as a result of t h e i r n e g a t i v e charge, s t a i n e d g r e e n only b y aniline green. T h e u p p e r a n d lower l i m i t s

of t h e isolelectrie a r e a of t h e t i s s u e s were c o m p a r e d w i t h m i x t u r e s of t h e a b o v e s t a i n s sealed
in glass a m p u l e s in p r o p o r t i o n 1 : 10, 1 : 9 a n d 9 : 1, l0 : 1. T h i s m a d e it possible to d e t e r m i n e t h e
u p p e r a n d lower l i m i t s of t h e isoelectric a r e a sufficiently a c c u r a t e l y a n d i n d i v i d u a l l y a t all stages.
I n w o r k i n v o l v i n g t h e d e s c r i p t i o n of t h e m e t h o d of a s c e r t a i n i n g I E P t h e r e is o f t e n no m e n t i o n
of t h e p u r i t y of t h e s t a i n s used. T h i s is a q u e s t i o n of first-rate i m p o r t a n c e a n d , therefore, we con-
sider it n e c e s s a r y to p a y s o m e a t t e n t i o n to it.
I n g e n e r a l m i c r o t e c h n i c a l practice t h e s t a i n s u s e d , e v e n t h e fairly pure, a l w a y c o n t a i n a cer-
t a i n a m o u n t of a d m i x t u r e . T h i s i m p u r i t y , w h i c h u s u a l l y possesses different qualities t h a n t h e
basic s t a i n , h a s a n u n f a v o u r a b l e effect o n t h e I E P v a l u e s o b t a i n e d . I t is t h e r e f o r e n e c e s s a r y as
a p r e l i m i n a r y m e a s u r e to verify t h e p u r i t y of t h e s t a i n s b y c h r o m a t o g r a p h paper. So, for e x a m p l e ,
acid f u e h s i n e c o n t a i n e d a c e r t a i n a m o u n t of basic a d m i x t u r e (probably basic fuehsine). K a t i o n s 9
o f t h i s a d m i x t u r e , a f t e r t h e I E P of t h e object u n d e r s t u d y is exceeded, t r a v e l b y eloctroad-
s o r p t i o n t o g e t h e r w i t h t h e k a t i o n s of t h e basic s t a i n (in o u r case aniline green). T h i s leads to a n
a p p a r e n t e x t e n s i o n of t h e isoelectric a r e a a n d t h u s also of I E P M v a l u e s t o w a r d s t h e a r e a of h i g h e r
pH.
W e r e m o v e d t h e u n d e s i r a b l e basic a d m i x t u r e f r o m acid f u c h s i n e b y a d s o r p t i o n w i t h active
charcoal r e p e a t e d several times. C h r o m a t o g r a p h i c control s h o w e d t h a t t h e acid f u c h s i n e w a s
n o w free of i m p u r i t y . T h e aniline g r e e n w h i c h we u s e d (imported, m a k e r n o t s t a t e d ) w a s v e r y
p u r e a n d f u r t h e r c l e a n s i n g w a s n o t required.
I E P M v a l u e s o f t h e objects m e n t i o n e d a b o v e were d e t e r m i n e d d u r i n g t h e period f r o m J u n e 11
to A u g u s t 6, 1957 a t r e g u l a r weekly intervals. V a l u e s e n t e r e d o n t h e g r a p h s were a l w a y s calcu-
lated s t a t i s t i c a l l y f r o m 4 s i m u l t a n e o u s o b s e r v a t i o n s . D e t e r m i n a t i o n of t h e significance of differ-
e n c e s w a s m a d e b y c a l c u l a t i o n of t h e v a l u e t. Significant differences are m a r k e d in t h e g r a p h s
b y t h e l e t t e r S.

30
2.5
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7 ~
i ~
A

2.5

2.5LI , ~ I l i L i L
,,16 ~816 2slG 21~, 91~' ~61~, ~31~' 3~I~ 618

F i g u r e 1. T h e values of I E P M of t h e g r o w i n g p o i n t s of t h e varieties of a p p l e T r a n s p a r e n t Yellow

(A), L o r d L a m b o u r n e (B) a n d W a t e r v l i e t (C).

Results and Discussion

The course of IEPM values of the growing points of buds showed two phases
during the period of study. In the first phase from the start to J u l y 7, 1957
I E P ~ values remained at the level IEPM 3.0 with maximum deviations of
0-1. In the second phase from J u l y 9 to August 8, 1957 IEPM values were of
quite a different character. With all three varieties there was a shift during
this period of IEPM values towards the area of higher p H (fig. 1). This shift
to the area of higher p H was only temporary and after a certain level was
reached (for Transparent Yellow 3.5, Lord Lambourne 3.6 and Watervliet
 I E P M VALUES OF GROWING POINTS OF F L O W E R BUDS 19

3.3) there was a shift of IEPM values back to the area of lower pH at the orig-
inal level. The most important fact, however, in this phase is t h a t in all
three varieties, during the period when, following the certain maximum ther~
was a backward shift of IEPM values to the area of lower pH, the beginning

s ~ s

./s lSla 251G z/~ 9/7 ~6/~, z~17 3~/~ ata

Figure 2. The values of I E P M of floem (I.) and xylem (II.) of brachyblasts of the varieties of
apple Transparent Yellow (A), Lord Lambourne (B) and Watervliet (C).

of the morphological differentiation of the growing points of flower buds was

recorded. Thus, in the case of Transparent Yellow the start of this process
was recorded on J u l y 23 and for the other varieties, Lord Lambourne and Water-
vliet, a week later on J u l y 31.
A similar tendency of IEPM values to shift towards the area of higher pH
was also observed in the case of phloem and xylem of brachyblasts and growing
spurs. The whole course of IEPM values for these tissues was further compli-
cated by the fact t h a t in the course of study it was found t h a t there were
two maxima of IEPM values in the area of higher pH (fig. 2 and 3). The first
maximum, which was not so marked as the second, was noted in the period
of the stoppage of growth in length in early summer (for Transparent Yellow
growth in length was halted on June 18 and for Lord Lambourne and Water-
vliet June 25). The second, more marked maximum of shift in value to the
area of higher pH was observed in the period of morphological differentiation
of the growing points of flower buds, t h a t is according to variety from J u l y
23 to J u l y 31, 1957.
The established fact of the shift in the I E P ~ values of meristematic tissues
of growing points in flower buds and of phloem and xylem of brachyblasts
and growing spurs, towards the area of higher pH offer us the opportunity to
fix beforehand the probable commencement of such an important pheno-
menon as the differentiation of growing points of flower buds.
20 B. HOI~AVKA

The question of whether this phenomenon will be analogous in different

years with our varying climatic conditions cannot at this period be answered
positively on the basis of our present knowledge. I t was, for example, found
t h a t the time difference in the start of morphological differentiation of the

Ii I , I I I I I
3"S

3"0 h

~ : ~ II
i

25

~7
30

2_5

I t ,
2/7 ~7 16/7 2~/7 31/7 6/a

Flgu,.e 3. The values of I E P M of floem (I.) and xylem (lI.) of growing spurs of the varieties of
apple Transparent Yellow (A), Lord Lambourne (B) and Watervliet (C).

growing points of flower buds for the same variety on one site is 14 days or
more. As a result we may expect a time shift of this and other phenomena
affecting this process both in the direction of extension and of reduction of
this time period. Definite differences can also be expected in varieties with
periodic fertility, particularly in years following a good crop, when the setting
of flower buds is minimal. In addition, the analogous course of IEPM value
changes in the vegetative and generative organs gives an indication of a mutual
relation existing between these organs.

References

TSEL1NIKER, J. L.: O fiziologlcheskoi differentsirovke tsvetochnikb pochek u jabloni. (On the

physiological differentiation of f e w e r buds of apple.) - - Dokl. A,N SSSI~ 66 : 281--284, 1949.
DRAWERT, H.: Das Verhalten der einzelnen Zellbestandteile fixierter pflanzlicher Gewebe gegen
saure und basische Farbstoffe bei verschiedenen W a s s e r s t o f f k o n z e n t r a t i o n e n . - Flora 1:12 :
91--124, 1937.
HO~AVXA, B.: Difereneiace kv6tnlch pupenfl jablonl. (Fruit-bud differentiation of apple trees.)
(~s. biologie (Praha) 6 : 462--465, 1957.
KONAREV, V. G.: Vozrastnie izmeneniya v kletkakh rasteniya i izoelektricheskaya toehka pro-
toplasmi. (Changes in plant cells during aging and the isoelectrie point of protoplasm.)
Dokl. AN SSSR 51} : 773~776, 1948a.
 IEP M VALUES OF GROWING POINTS OF FLOWER BUDS 21

KONAREV, V. G. : V o z r a s t k l e t o k r a s t e n i y a ~ o t n o s h e n i y e k l e t o c h n i k h o b e l o c h e k k kislim i osnov-

h i m k r a s k a m . (The age of p l a n t ceils a n d t h e relation of cell walls to acidic a n d basic stains.)
Dokl. A N S S S R 59 : 9 8 3 - - 9 8 6 , 1948b.
SC~WA~TnS, I t . O.: F / ~ r b u n g s a n a l y t i s c h e U n t e r s u c h u n g e n z u r L a g e des i s o e l e k t r i s c h e n P u n k t e s
d e r Z e l l b e s t a n d t e i l e in w a e h s e n d e n Zellen u n d Geweben. - - P r o t o p l a s m a 41 : 3 8 2 - - 4 1 4 , 1952.
ST~UGGE~, S.: l~ber d a s V e r h a l t e n des pflanzliehen Zellkerns gegeniiber Anilinfarbstoffen. - -
P l a n t a 18 : 5 6 1 - - 5 7 0 , 1932.
Z E I O ~ , K . : D e r E i n f l u s s y o n F i x i e r u n g s m i t t e l n a u f dio F/~rbbarkeit histologischer E l e m e n t e . - -
Z t s c h r . wiss. Mikrosk. 47 : 273:--293, 1930.

Address: Bo}ivoj I-Io~avka, I n s t i t u t e of Biology Czechoslovak A c a d e m y of Sciences, N a cviSi~ti 2,

Praha-Dejvice.

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