Indian J Plant Physiol., Vol. 6, No.4, (N.S.) pp. 355-360 (Oct.-Dec.

, 2001)

PACLOBUTRAZOL MODIFIES TREE VIGOUR AND FLOWERING IN MANGO

CV. ALPHONSO
G.S.R. MURTII., K.K. UPRETP, R.M. KURIAN3 AND Y.T.N. REDDy4

Plant Hormones Laboratory 1.2, Fruit Culture Laboratory 3.4, Indian Institute of Horticultural Research, Hessaraghatta Lake Post,
Bangalore 560 089

Received on 9 Jan., 2001

SUMMARY
Cumulative effects of three annual soil drenching treatments ofpaclobutrazol (5 and 10 a.i. g/tree) given
to the mango cv. Alphonso during months of Aug-Sept revealed reductioQ in tree height, mean shoot length and
number of dormant shoots, and enhancement in flowering. Between the treatments, 109/tree paclobutrazol
treatment was more effective in altering the vigour determining morphological characters. Flowering intensity
did not differ much between the two treatments. The anatomical features under paclobutrazol treatment (lOg
a.i.ltree) revealed higher phloem to xylem ratio; more so in the shoots of current year flush than the previous
year ones. Xylem sap volume declined with the paclobutrazol treatment. The paclobutrazol treated trees
responded with an enhancement in the levels of abscisic acid (ABA) and cytokinin-zeatin riboside (t-ZR) and
dihydtozeatin riboside (DHZR) in the xylem sap and leaves, and phenols in leaves concomitant with the decline
in leaCindole acetic acid (IAA) content; changes being greater under higher dose ofpaclobutrazol. These results
show that the paclobutrazol induced inhibition in tree vigour and promotion of flowering in mango is not only
associated with the gibberellin biosynthesis inhibiting character ofpaclobutrazol but also with its influence on
other hormones such as ABA, cytokinins and IAA and as also on phenol.

Key words: ABA, cytokinins, IAA, mango, paclobutrazol, phenols, stem anatomical features, xylem sap

INTRODUCTION response of paclobutrazol has been revealed as the

In the high density plantation offruitcrops, controlling
tree vigour and canopy size are important for enhancing
the orchard efficiency and productivity. Out of the several
strategies suggested, use of dwarfing rootstocks (Turkey
1964) and growth retardants (Davies and Curry 1991,
Fletcher et ai., 2000) have been found to modify growth
and development and to increase yield in a numberoffruit
crops including mango. Amongthe growth retardants, the
soil drenching with triazole compound - paclobutrazol,
prior to flower bud differentiation has been reported to
inhibit vegetative growth and enhance flowering in mango
(Kulkarni 1988, Burondkar and Gunjate 1993, Kurian
and Iyer 1993, Murti and Upreti 2000). This inhibitory
consequence of inhibition in the biosynthesis of elongation
growth promoting hormone gibberellins, known to be
synthesized following isoprenoid pathway. The isoprenoid
pathway besides synthesizing gibberellins, also partially
regulates the synthesis of other important endogenous
hormones such as abscisic acid (ABA) and cytokinins.
Considering that the plant growth is regulated by
interaction among endogenous hormones and the levels
of one ~ormone influence the level of others, the growth
inhibitory response of paclobutrazol could better be
explained by changes in the levels of different hormones
rather than single hormone. There are limited evidences
that endogenous hormonal levels mediate the tree vigour
regulation in mango by paclobutrazol application.

• Corresponding author, E-mail address: gsrm@iihr.kar.nic.in

IndianJ. Plant Physiol., Vol. 6, No.4, (N.S.) pp. 355-360 (Oct.-Dec., 2001) 355
 G.S.R. MURTI et al.
Attempts have thus been made to elucidate the role of
endogenous hormones such as ABA, cytokinins-zeatin
riboside (t-ZR) and dihydrozeatin riboside (DHZR) and
indole acetic acid (IAA), and phenols in relation to
paclobutrazol-induced reduction in tree vigour and canopy
size in mango cv. Alphonso.
MATERIALS AND METHODS
The experiment was conducted at the mango orchard
of the Ind ian Institute of Horticu Itural Research,
Hessaraghatta. The J 7 yearold mango trees ofcv. Alphonso
were given three annual soil drenching treatments of
paclobutrazol at 5 and 109 a.i. per tree during the months
of August-September (period before flower bud
differentiation) in the years J 996, J 997 and J 998. After
each treatment, the trees were irrigated sufficiently for
dispersion of the growth regulator into the soil.
Observations on morphological attributes such as
tree height, tree volume, trunk girth, shoot length and
percentage of flowering, vegetative and dormant shoots
were recorded in 1999. Simultaneously, uniformly
growing vegetative shoots of current and previous year of
growth, and leaves from previous year flushes were
collected both from paclobutrazol- treated and untreated
trees. Stem anatomical features were recorded in both
types of shoots, while shoots of previous year were used
for xylem sap extraction. The xylem sap and leaves were
used fordetermining the changes in endogenous hormones.
Total phenol content was estimated on Iy in leaves. Xylem
sap was extracted under reduced pressure in the vacuum
flask and its volume was recorded. For stem anatomy,
stem segment (5mm thickness) from four shoots each
from treated and untreated trees were cut. The stem
segments were fixed in the fixative solution comprising
offormalin: acetic acid: ethyl alcohol: water (J 0:5 :50:35
v/v). These were then dehydrated in various alcohol
dilutions (70 to 100%), treated further with xylene and
embedded in paraffin wax according to Jensen (1962).
The serial sections of shoot segments (15 ~m) were
mounted on 1% (w/v) gelatin-precoated sl ides. The xylem
and phloem tissues were observed after staining with
safranine (0.5%, w/v) in 50% (v/v) alcohol, and radial
width of primary xylem from inner edge of vascular
bundles to cambial ring and of primary phloem from
cambial ring to outer periphery of vascular bundles was
356
determined employing a calibrated microscope (x 100
and x 60 magnification for current and previous year
shoots, respectively). For hormonal estimations, the leaf
samples (5g) were extracted in chilled 80% (v/v) aqueous
methanol. After filtration ofmethanolic extract, the filtrate
was concentrated in vacuo at 35°C, residue dissolved in
water and pH adjusted to 3.0. The acidic extract (2g
equivalent) was partitioned thrice with chilled diethyl
ether, and the ether phase evaporated. The residue was
taken in 1.0 ml of 20 mM tris buffer pH 7.5 containing
5mM MgCI2 and 20mM NaN) for ABA and IAA
estimation. The remaining 3.0 g equivalent extract was
used for analyzing cytokinins (t-ZRand DHZR). Forthis,
acidic extract (3.0 g equivalent) was partitioned thrice
against water saturated n-butanol after adjusting the pH to
8.2. The butanol phase collected was evaporated in vacuo
at 35°C and the residue was taken up in above tris buffer
(1.5 ml). The xylem sap was used as such forthe estimation
of different endogenous hormones. The estimations of
ABA (Weiler 1982), cytokinins- t-ZR and DHZR(Barthe
and Stewart, 1985) and IAA (Weiler et al., 1981) in the
leaves and xylem sap were performed by the ELISA
technique employing the laboratory raised polyclonal
antibodies. Total phenol content in tht< leaves was
determined according to Cole (1984) using chlorogenic
acid (Sigma) as the standard.
RESULTS AND DISCUSSION
Morphological characters: Paclobutrazol treatments
considerably altered the tree height, tree volume, shoot
length, num bel' of dormant shoots and flowering in mango
cv. Alphonso (Table 1). The 109 a.i/tree treatment
resulted in reduction in tree height (21.2%), tree volume
(33.1 %)and mean shoot length (48.2%). The shoots from
paclobutrazol treated trees flowered profusely (76.2%)
with marked reduction in the number of dormant shoots
(J 8 fold) under 5g a.i/tree treatment. However, the
differences between the two paclobutrazol treatments
with respect to these attributes were small (Table 1). The
percentage of vegetative shoots was unaltered under 10g/
tree paclobutrazol treatment as compared to lower level
of paclobutrazol. Thus paclobutrazol increased the
percentage oftlowering shoots and also inh ibited extension
growth of vegetative shoots and tree spread. This response
ofpaclobutrazol is attributed to GA-inhibiting activity of
Indian J. Plant Physiol.. Vol. 6, No.4, (N.S.) pp. 355-360 (OCI.-DeC., 2001)
 PACLOBUTRAZOL INDUCED CHANGES IN ALPHONSO MANGO

Table 1. Effect ofPaclobutrazol on tree vigour and flowering in mango cv. Alphonso

Treatments Tree height Tree volume Trunk girth Mean shoot % Shoots
(m) m3 (em) length (em) Flowering Vegetative Dormant

Control 5.90 210.2 112.5 22.6 52.5 2.5 45.0

Paclobutrazol 5.50 195.9 111.5 15.9 92.5 5.0 2.5

(5g a.i/tree)

(10g a.i/tree 4.65 140.6 103.5 11.7 95.0 2.5 2.5

Data represent mean of replications

paclobufrazol as gibberellins are known to promote
elongation growth and Inhibit flowering in mango (Tomer
1984).
Anatomical features: Stem anatomical features
between the paclobutrazol-treated and untreated trees
showed marked differences in the differentiation pattern
in the shoot tissues (Table 2). There was reduction in
xylem radial width and increase in that of phloem in the
shoots from both current and previous year flush under
paclobutrazol treatment. The reduction in xylem radial
width was greater in the shoots of current year flushes
(37.0%) than the previous year ones (4.7%), while increase
in phloem radial width was greater in the shoots of
previous year flush (27.6%) than the current year ones
(9.0%). The ratio of radial widths of ph loem to xylem also
varied significantly in both the types of shoots, with the
ratio being high in the current year shoots (Table 2). The
paclobutrazol induced decline in xylem tissues and increase
in phloem tissues is in line with the observations of
Aguirre and Blanco (1992). Also the findings of Kurian
and Iyer (1992) that the higher ratio of radial width of
phloem to xylem tissues is associated with dwarfing
nature in mango trees further lend support to our results.
Xylem sap: Xylem sap volume from the shoots of
paclobutrazol-treated trees was lower than that from
untreated ones (Table 3). The 109 a.i/tree paclobutrazol
treatment resulted in 37.0% decline in sap volume over
untreated trees. This reduction in xylem sap volume in the
paclobutrazol treated shoots could be the result of
thickening of xylem vessels as evident from anatomical
changes. Also the xylem sap is an effective carrier fluid
for essential soil nutrients and other root produced growth
hormones to different plant organs. Thus the lowering of
sap yield in the shoots by paclobutrazoI would be important

Table 2. Effect ofpaclobutrazol on stem anatomy in mango cv. Alphonso

Treatments Radial length (j.1m)

xylem Phleom Phleom/xylem ratio

Control Current year 50.67 36.37 0.72

(±2.05) (±1.09)
Previous year 161.29 43.81 0.27
(±6.16) (±2.18)

Paclobutrazol Current year 31.92 39.65 1.24

( 109 a.iltree) (±1.47) (±0.95)
Previous year 153.65 55.88 0.36
(±4.27) (±2.85)

Values in the parentheses are standard error of mean

Indian J. Plant Phy,iol .• Vol. 6, No.4, (N.S.) pp. 355-360 (Oct.-Dec., 2001) 357
 G.S.R. MURTI et al.

in imparting the tree growth suppression. However, this
aspect needs further investigation on sap flow.
Endogenous hormones: Paclobutrazol treatments
distinctly influenced the levels of endogenous hormones
both in the xylem sap as well as leaves (Table 3). The
general trends with respect to hormonal changes were
however, similar both in xylem sap and leaves. The ABA
content increased by 55.5 and 114.5% in the leaves and
132 and 214.5% in the sap, respectively, under 5 and 10
g a.i/tree paclobutrazol treatments. Jindal et at. (1974)
reported that the shoot growth retardation in apple is
accompanied with enhanced levels of ABA. Kurian et al.
(1991) reported sl ightly lower levels of ABA in a dwarf
cultivar as compared to vigorous cultivar and also in
paclobutrazol treated plants as compared to untreated.
They had, however, observed a three-fold increase in
ABA in dormant buds as compared to active ones. The
present study differs in the tissue analyzed, the levels and
number of applications employed, season of drawing
samples and the quantification technique. Murti and
Upreti (1999) in their studies on two cultivars of mango
differing in tree vigour reported higher ABA and lower
IAA levels during active shoot growth responsible for
vigour restriction in mango. The enhancement in ABA
levels under paclobutrazol treatments could be the result
of alteration in its metabolism. There are no evidences in
support of modification of metabolism under triazole
compounds treatment. However, possibi Iity of changes in
ABA levels via alteration in metabolism cannot be ruled
out as ABA shares isoprenoid pathway similar to
gibberellins for ABA synthesis. Hauser et al. (1990) on
the other hand suggested that increase in ABA levels by
triazole compounds is due to prevention of its catabolism
to phaesic acid.
There was increase in the content of cytokinins- t-ZR
by 25.7 and 53.0% in the leaves and 10.3 and 70.3 in the
xylem sap and in DHZRcontent by 38.1 and 96.3 % inthe
leaves and 32.3 and 54.6% in the sap, respectively, under
5 and 10 g a.i.Itree paclobutrazol treatments (Table 3).
The t-ZR and DHZR are important translocational forms
of growth promoting cytokinins. The increase in these
ribosyl derived cytokinins is reported to act positively in
flower bud formation (Izumi et al. 1988). Chen (1987)
and Agrawal et al. (1985) suggested that the increased
cytokinins are assodated with flower bud induction in
mango. Also similarto this, the exogenous applications of
cytokinin-BA have been found to accelerate flower bud
formation in mango (Chen 1985) and apple (Bruinsma
1979). However, it seems unreasonable to account for
such physiological effect of paclobutrazol solely to
enhancement in cytokinins as decline in gibberellins also
favours flowering. The changes in cytokinins as observed
in the present study are at variance with that reported by
Kurian etal. (1992). Using bioassay techn ique employing
soybean cotyledon callus hypocotyl, they reported higher
levels ofZR/DHZR, Z/DHZ and cytokinin glucoside like
compounds in the leaves ofpaclobutrazol-untreated trees
than the treated ones. This discrepancy could be due to the

Table 3. Changes in xylem sap volume and endogenous hormones in h. '-ition to paclobutrazol treatment in mango
,cv. Alphonso

Treatment Sap Voi/tO ABA t-ZR DHZR fAA Phenolic

content
Shoots (ml) leaf (ng/g) sap (ng/ml) leaf (nglg) sap (nglml) leaf (ng/g) sap (ng/ml) leaf (nglg) (ng/g)

Control
0.73 23.5 30.4 44.0 63.0 59.0 104.0 48.9 7.9
(±0.17) (±1.18) (± 1.35) (±2.36) (±2.86) (±3.57) (±3.97) (±2.21) (±0.13)

Paclobutrazol
(5g a. i/tree) 0.72 36.6 70.7 55.3 69.5 95.3 137.5 25.2 10.6
(±0.04) (±1.73) (±3.08) (±2.82) (± 1.98) (±1.06) (±1.84) (±1.08) (±0.61)

(lOg a.i/tree) 0.46 50.4 95.6 67.3 107.2 115.8 160.8 19.1 12.9
(±O.02) (± 1.52) (±2.73) (±1.56) (3.29) (±5.18) (±5.18) ( 1.63) (0.48)

Values in the parentheses are standard error of mean

358 Indian J Plant Physiol., Vol. 6. No.4, (N.S.) pp. 355-360 (Oct.-Dec., 2001)
 PACLOBUTRAZOL
differences in the methodology for estimation of
cytokinins. The separation methods and the quantification
based on bioassay are no longer considered precise. The
bioassays lack specificity and are much less sensitive as
compared to the recent techniques such as ELISA.
The IAA could be detected in the leaves only. Its
content showed a decline of 48.5% under 5g a.i/tree
treatment and 60.9% under 109 a.i/tree treatment (Table
J). There are reports showing gibberellins as promotory
to IAA biosynthesis (Law 1987). The lowering ofIAA by
paclobutrazol treatments could be related to inhibition in
gibberell in biosynthesis, known to occur by paclobutrazol.
Thus the effect of paclobutrazol on IAA may not be direct,
but could be mediated by its effect on gibberellins. Law
and Hamilton (1989) reported that the reduction in
internodal length by triazole compound-uniconazole is
accompanied by reducing levels ofIAA. Also, the studies
of But a et al. (1989) showing higher levels ofIAA in the
leaves of vigorous apple trees in the early and late growing
season further strengthen our findings.
Total phenols: Total leaf phenol content in the
paclobutrazol-treated trees was higher than the untreated
trees (Table 3) by 34.2 and 63.3% in 5g a.i/tree and 10 g
a.i/tree paclobutrazol treatments. Thus the effect of
paclobutrazol on the phenol content is similar to that on
ABA. Kurian et al. (1994) reported that the phenol
content in mango is inversely related to tree size and
vigour. Thus paclobutrazol induced downward regulation
of tree vigour could be the result of enhancement in
phenol content. Further, vigour of a tree is related to cell
division (CD) and cell extension (CE). Higher phenol
content with lowered IAA levels in a tissue would supress
the tree growth by influencing CEasIAAand gibberellins
are known to promote extension growth. The higher
phenol is also reported to affect extension growth by
lowering IAA levels as a result of induction in IAA-
oxidase activity (Zenk and Muller 1963).
The paclobutrazol induced inhibition in tree vigour
and promotion offlowering in mango is not only associated
with gibberellin 'inhibiting property ofpaclobutrazol, but
also to its influence on conducting tissues and levels of
endogenous hormones, ABA and cytokinins - t-ZR and
DHZR in xylem sap and leaves, and IAA in leaves
Indian 1. Plant Physiol., Vol. 6, No.4, (N.S.) pp. 355-360 (Oct.-Dec.,
INDUCED CHANGES IN ALPHONSO MANGO
together with leaf phenol content. However, it needs to be
further explored whether the changes in above parameters
are the consequence of inhibition in gibberellin
biosynthesis or are the direct influence of paclobutrazol.
ACKNOWLEDGEMENT
The authors are thankful to the Director ofthe institute
for providing necessary facilities and toNr. H.L. Jayaram
and Mr. K.R. Earanna for technical assistance.
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