J ournal of Experimental Sciences Vol.

2, Issue 2, Pages 16-20 [2011]

www.jexpsciences.com
* Corresponding Author, Email: [email protected], Mob: +91 9789344418 J ES
ISSN: 2218-1768

Regular Article
Thermal Sensitivity and Sucrose Accumulation in Response to Chemical
Ripener Application in Sugarcane

K. Shanmugaraja
1*
, M. Vigneeswaran
1
and S. Venkataramana
1,2


1
Department of Biotechnology, Ayya Nadar J anaki Ammal College, Sivakasi.Venture institute
of biotechnology Madurai;
2
Department of Plant Physiology, Sugarcane Breeding Institute,
Coimbatore-641007
ABSTRACT: Effect of two promising chemical ripeners (Ethrel and
Glyphosate) was tested on the sucrose accumulation process of two
popularly grown sugarcane varieties (Co 86032 and Co 94012)
during maturity and ripening phase (240 days to 360 days).
Invertase enzymes (acid and neutral invertase) responsible for
sucrose accumulation, electrolyte leakage in relation to
temperature, hormonal analysis through HPLC, sucrose % and Brix
% of juice were studied in ethrel or glyphosate treated cane
varieties. The acid and neutral invertases increased with moderate
increase in temperature. However, 50 C treatments were inhibitory
for acid invertase particularly in Co 86032 at 360 days. At final
harvest the sucrose% juice was 19.22% in Co 86032 and 21.98% in
Co 94012. Ethrel has shown 0.7% improvement in Co 86032, while
glyphosate treatment had shown 4.59% improvement in sucrose%
juice.

Key words: Sugarcane, Ethrel, Glyphosate, Sucrose accumulation,
Invertase enzyme
Introduction
Sugarcane (Saccharum officinarum, L. commercial hybrid), an
annual crop of tropics is an important life sustaining crop cultivated
in both tropical and subtropical climates. The crop passes through
four distinct physiological growth phases. The first phase i.e.
germination phase (0-60 days) denotes activation and sprouting of
vegetative bud. The germination is followed by the formative phase
(60 150 days) during which profuse tillering is initiated. The third
important growth phase is the grand growth which starts around
150 days and ends by 240 days. During this phase the stalks grow
rapidly almost at the rate of 4-5 internodes / month under favorable
conditions. The grand growth is followed by maturity and ripening
phase (240 to 360 days) during which the accumulation of sucrose
in the cane stalk (Van Dillewijn, 1952) is at maximum. Sucrose
accumulation is regulated by invertase enzymes which have been
suggested to be the key regulators for accumulation of sucrose in
parenchyma and also play a vital role in cane elongation and
maturation of sugarcane stem. Two soluble invertases viz., acid
invertase and neutral invertase along with cell wall bound acid
invertase are known to be present in sugarcane storage tissue. Acid
invertase is positively correlated with cane elongation, while neutral
invertase is related to sucrose content. The acid invertase occurs in
particulate cell membrane or in the cell wall free space. The neutral
invertase is a soluble enzyme and hydrolyses sucrose at moderate
levels (Zhu et al., 1997). Moore (1987) accounted that the
sugarcane varieties are capable of storing sucrose in parenchyma
tissue of the stem up to 26%. The modern sugarcane hybrids have
the potential of accumulating about 22 to 23% sucrose in juice.
Ripening can be enhanced by drying off cane (Robertson and
Donaldson, 1998), and by lowering of temperature (J ames, 1999).
Artificial ripening of sugarcane has been made possible by the use of
chemical ripeners that hasten sugarcane maturation and increase
sugar yield (Nickell, 1984). Ripeners are chemical agents used to
induce the ripening. Most chemical ripeners are used to increase the
sucrose content at harvest in many countries like Australia, South
Africa, Guyana, Swaziland, Hawaii, Mauritius, Florida and Louisiana
and in Brazil. In order to study the influence of two chemical
ripeners(Ethrel or Glyphosate), a field experiment was conducted
during 2009-2010 crop season at Sugarcane Breeding Institute,
Coimbatore, utilizing two popularly cultivated sugarcane varieties
namely Co 86032 and Co 94012. These chemicals were sprayed at
200 ppm and 400 ppm concentration at the maturity initiation (240
days), and at 300 days.

Materials and Methods
A field experiment was conducted at Sugarcane Breeding Institute,
Coimbatore (76.59 E longitude and 11.02 N latitude, 426.72 m
TMSL), India, during 2009-2010, utilizing two sugarcane
(Saccharum officinarum, L.) varieties Co 86032 and Co 94012
representing high sugar content at maturity. Healthy and disease
free planting material was taken from 10 month old crop, and two
bud cuttings taken from the middle and top portion of cane were
planted (40 per row of 6.0m length, spaced 0.9m apart) using split-
plot design replicated thrice. The gross and net plot sizes were
32.7m
2
and 21.8 m
2
, respectively. The soil (Typic Haplustalf, Noyyal
series) is of a sandy loam type and the fertility status is low: high:
high with respect to N: P: K. the crop was fertilized with Urea, P2O5
and K2O (N: P: K) at 225:65:120 kg/ha. Nitrogen (Urea) and potash
(K2O) were given in two split doses at 45 and 90 days age and P
(Super phosphate) was applied at the time of planting.

Ripener application
During maturity phase (240-360 days age) the chemical ripeners
(Ethrel or Glyphosate) were sprayed in split doses, i.e. at the
commencement of maturity initiation (240 days) and at 360 days
age. Ethrel or Glyphosate at 200 ppm and 400 ppm were prepared
using commercial grade ethrel (39% a.i.) and glyphosate (41% a.i.).
The chemicals were diluted with water and a few drops of surfactant
(Triton X-100) were added to facilitate the entry of the applied
chemicals. Three undisturbed rows in each set of treatment were
sprayed with either of these two chemicals up to the full drenching
of the top canopy. Water spray served as controls.

Acid and Neutral Invertase enzymes
Acid and neutral invertases were assayed at monthly intervals during
maturity initiation (240 days) and at full maturity (360 days). A stem
sample of each variety from each replication was cut, stripped and
topped. The rind portion of stem was removed and different
temperature treatments (30C, 40C, and 50C) were given. A
control was maintained where the stem tissue is kept at room
temperature. Uniform discs weighing 1.0g were taken from the
pooled top, middle, and bottom internodes of cane for the assay of
acid or neutral invertase enzymes. The stem discs were treated with
5ml of cold ethyl acetate for 20 minutes and washed with distilled
water until the smell goes off. Invertase activity was estimated in a
reaction mixture (10ml) containing 0.05M citrate buffer (pH 5.0) for
acid invertase or 0.05M sodium phosphate buffer (pH 7.0) for
neutral invertase, 0.2M sucrose, distilled water and 0.1 ml of toluene
along with stem discs. The tubes were incubated at 30C for 30
minutes. An aliquot of 0.5 ml of the reaction mixture was taken to
which 1ml copper reagent was added and the tubes were kept in
water bath for 10 minutes and then cooled. The reducing sugar
content was estimated using arsenomolybdate reagent, and the
absorbance was read at 700nm using a UV-VIS Spectrophotometer
(Shimadzu, J apan). The amount of reducing sugar formed was
quantified against a standard curve prepared with known
concentration of glucose in identical conditions.
J Exp Sci Vol. 2, I ssue 2, Pages 16-20 [2011]



Cellular membrane thermostabilty
Young and actively growing leaf from the main shoot of the plant
was cut, cleaned under running tap water, blotted dry. Leaf discs
(0.5cm diameter) weighing 0.2g were cut(free from mid vein) and
infiltrated in 10 ml distilled water in a test tube, and the initial
electrical conductivity (Eca) was measured using a conductivity
meter. The tissues were incubated in water bath (35C, 45C, and
55C) for 30 min, and the electrical conductivity (Ecb) was recorded.
Subsequently the tissue was incubated in boiling water bath (100C)
for 10 min and the electrical conductivity (Ecc) was measured.
Membrane damage was calculated using the following formula.
% Electrolyte leakage = Eca-Ecb x100 / Ecc

Hormone extraction
The endogenous level of hormones in the shoot apices of stalk of
clones Co 86032, Co 94012 were quantified using HPLC (Shimadzu
VP- series). Twenty grams of shoot tips were weighed and
macerated with 70% methanol along with 2% ascorbic acid in a pre
chilled mortar and pestle. The standard procedures were followed
for the extraction of hormones in shoot apices as per Kuhnle et al,
(1983). The basic, acidic, and bound form of hormones were
fractionated and combined in HPLC Grade methanol for HPLC
analysis.

HPLC Analysis
Prior to use, the methanol fraction of the solvent was filtered
through a membrane filter and degassed. The sample was run on a
reverse phase C18 column with acetonitrile: water (1:3) as the
mobile phase and the chromatogram was obtained at the flow rate
of 1.0 ml/min and read in UV-VIS detector at 254 nm. The retention
time (RT) for hormones was observed at 5-15 min. the identification
of hormones was done by comparison of retention time with
authentic standards of hormones (Sigma). The concentration of the
hormones in plant samples was calculated using the peak area
against the hormones standard values and expressed as g/g.

J uice analysis
Sucrose %
The fresh cane juice was clarified by adding lead acetate and filtered
through Whatman filter paper No: 1. the sucrose % juice was
quantified through Polarimeter.

Brix %
Total solids present in the juice were expressed in percentage. The
brix% juice was quantified through Polarimeter.

Results and Discussion
Acid invertase
At 30 C, in both the varieties, the acid invertase activity was
reduced at 200 ppm and a marginal increase was seen at 400 ppm
in response to ethrel or glyphosate. At 40 C under ethrel treatment
in, the enzyme activity was increased at all levels. Under glyphosate
treatment (200 ppm), the activity was reduced while increased at
400 ppm in both varieties (Fig. 1). At 50 C the average enzyme
activity was 3.02 mol gfrwt
-1
h
-1
in Co 86032, and 2.94 mol g frwt
-
1
h
-1
in Co 94012 under ethrel treatment. Under glyphosate treatment
the average activity was similar in both the varieties.


Fig.1. Acid Invertase activity at different temperatures in response to chemical ripener application













Neutral invertase
At 30 C, the neutral enzyme activity decreased at all levels of ethrel
treatment in Co 94012. In Co 86032 at 200 ppm the activity was
reduced (2.08 mol gfrwt
-1
h
-1
) but increased at 400 ppm (2.78
mol gfrwt
-1
h
-1
) as compared to control (2.37 mol gfrwt
-1
h
-1
).
Under glyphosate, in Co 94012, the activity was increased at 200
ppm and declined at 400 ppm. At 40 C under ethrel treatment, in
both varieties, the neutral invertase activity declined as compared to
control. Glyphosate at 400 ppm increased the activity in both
varieties (Fig.2). At 50 C treatment, in Co 94012, the activity was
increased in 200 ppm ethrel (3.10 mol gfrwt
-1
h
-1
) and as well as in
400 ppm also (3.24 mol g frwt
-1
h
-1
) as compared to control (2.47
mol gfrwt
-1
h
-1
). Under glyphosate treatment the activity was
decreased at 200 ppm but increased at 400 ppm in both the
varieties.


Fig.2. Neutral invertase activity at different temperatures in response to chemical ripener application















Cellular membrane thermo stability
The electrolyte leakage at 35 C under ethrel treatment showed a
decline of 9.99 % at 200 ppm and 14.83 % at 400 ppm in variety Co
86032. In Co 94012, a slight improvement was noticed at all levels.
Under glyphosate treatment in Co 86032 the leakage was 28.28 %
at 200 ppm and 13.02 % at 400 ppm. At an elevated temperature of
45 C, in Co 86032, under both chemical treatments the leakage
was comparable with control (60.72 %). In Co 94012, glyphosate
treatment (400 ppm) showed high leakage (46.42 %) as compared
to 200 ppm (21.23 %) and control (5.40 %). At 55 C under ethrel
treatment(200 ppm), the leakage was 9.99 % as compared to the
control (24.26 %) in Co 86032, and at 400 ppm the electrolyte
J Exp Sci Vol. 2, I ssue 2, Pages 16-20 [2011]


leakage was high (25.82 %). The electrolyte leakage was high in Co
94012 in response to both the chemicals. The linear increase in
temperature treatment has influenced the leakage of cell contents in
the leaf tissue. The cell leakage increased gradually with increase in
the temperature (Fig.3). The maximum electrolyte leakage was
observer at 45C in both varieties under chemical ripener treatment,
thus suggesting the vulnerability of leaf tissue to elevated
temperature treatment and the consequent damage to the cellular
membrane.


Fig. 3. Membrane injury% at different temperatures under the influence of ethrel or glyphosate















Hormones
Using HPLC, hormone content in meristem was estimated in both Co
86032 and Co 94012 in response to the ripener application during
the maturity phase. The hormones were quantified against Indole
Acetic Acid (IAA) and Gibberellic Acid (GA) which served as the
standards.
In Co 86032 (control) the gibberellic acid and indole acetic acid
showed peaks having an area percent of 12.70 % and 4.82 % at
retention time (RT) of 5.97 min and 15.45 min, respectively. In
response to 200 ppm ethrel, the IAA peak showed maximum area
percent of 10.23 % and the GA showed an area percent 2.95 % at
RT of 5.96 min and 15.35 min, respectively(Fig.4). In 400 ppm
ethrel, maximum area percent of 8.29 % in IAA at the RT of 15.36
min was noted, but the GA area percent was 4.35 % and the RT
was 5.98 min. Under glyphosate treatment at 200 ppm the GA and
IAA peaks had an area percent of 5.11 % and 4.19 % at RT of 5.98
min and 15.39 min, respectively. At 400 ppm, the GA and IAA peak
had an area percent of 8.73 % and 7.50 % respectively. The RT of
both hormones was 5.97 min and 15.37 min, respectively.
In Co 94012 under ethrel treatment the control plants had the GA
and IAA peaks possessing the area percent of 7.89 % and 8.10 % at
RT of 5.97 min and 15.50 min, respectively. At 200 ppm, both GA
and IAA peaks resulted in an area percent of 5.08 % and 9.66 % at
RT of 5.99 min and 15.52 min, respectively. The results showed that
at 400 ppm, the GA area percent was 4.86 % at a RT of 6.01 min.
The IAA area percent was 7.34 % at RT of 15.52 min. Under
glyphosate (200 ppm) treatment, the GA and IAA peak area
percents were 5.70 % and 5.89 % at RT of 6.02 min and 15.65 min,
respectively. At 400 ppm the GA peak area percent was 6.42 % at
RT of 6.01 min, while IAA peak area percent was 7.63 % at RT of
15.62 min. Many minor peaks at different retention times were
noticed at all stages and only GA and IAA peaks only were
prominent.


Fig. 4. Hormone content in sugarcane during maturity and ripening phase

















Pk# Name Retention Time Area Area % Height Height %
9 GA 5.97 1164513 12.70 72438 14.99
23 IAA 15.45 442232 4.82 12824 2.65









Pk# Name Retention Time Area Area % Height Height %
7 GA 5.97 2569456 7.89 144213 10.87
22 IAA 15.50 2639770 8.10 71251 5.37
J Exp Sci Vol. 2, I ssue 2, Pages 16-20 [2011]



The hormone analysis through HPLC revealed the formation of
various peaks at different retention times. Application of GA was
known to increase the stalk fresh weight, total stalk length and the
length of individual internodes. Earlier reports showed that
Gibberellins (A1, A3, A4, A19, A20 and A36) were identified by gas
chromatography selected ion monitoring (GC-SIM) in apices of
sugarcane. GA3 increases the growth of cane which finally helps
improve the yields.
Gibberellins are also known for promoting cell growth through
increased hydrolysis of starch and sucrose into glucose and fructose
molecules. These hexoses provide energy via respiration and
contribute to cell wall formation. As a result of the decrease in water
potential, water enters more rapidly, causing cell expansion but
diluting the sugars. In sugarcane stems gibberellin- promoted
growth results in part from increased synthesis of invertase enzymes
that hydrolyze incoming sucrose to glucose and fructose. Since,
gibberellins are responsible for cell elongation process, in the
present study also, the GA3 were prominently present in both
varieties.
The endogenous hormones particularly GA3 and IAA might
contribute for elongation of stem and cane growth i.e., higher level
of GA3 and lower level of IAA favors better stem growth particularly
in Co 97008, Co 86032, Co 99004 and Co 2000-10. Also level of GA3
and IAA might play a prominent role to mitigate the stunted growth
in ratoon crop. I n sugarcane genotypes with higher levels of IAA
influence the tiller production (Vasantha et al., 2004).

Sucrose% juice
Sucrose is the most important product in sugarcane. Under ethrel
treatment, the average sucrose % was from 16.32 % at 240 days
which has gone up to 19.12 % at 360 days in Co 86032(Fig.5). At
200 ppm treatment, the sucrose % showed a slight improvement
and at 400 ppm at harvesting stage, sucrose% improved by 0.7%
as compared to control in Co 86032. In Co 94012 the average
sucrose % was from 17.38 % at 240 days which has reached to
21.79 % at 360 days. The maximum amount of sucrose % was in
Co 94012 under ethrel (200 ppm) treatment (22.95 %). Under
glyphosate treatment in Co 86032 the average sucrose % increased
from 15.50 % at 240 days to 19.66 % at 360 days. At harvesting
stage, the maximum sucrose % was 22.53 % in Co 94012.


Fig. 5. Sucrose % at maturity phase in response to chemical ripener application
















Brix %
Brix (total solids) estimated at monthly intervals during maturity
showed gradual improvement in both varieties. Under ethrel
treatment, the average brix % increased from 18.73 % at 240 days
to 23.57 % at 360 days in Co 86032(Fig.6). But in Co 94012 at 200
ppm in 270 days the brix % was 20.55%, it was slightly declined
(0.05 %) as compared to 240 days brix (20.60 %). In Co 86032
under ethrel treatment the brix % was slightly declined from control.
Under glyphosate treatment, average improvement was in Co 86032
from 17.92 % at 240 days to 24.21 % at 360 days. Similarly in Co
94012 the brix improved from 19.01 % at 240 days to 22.76 % at
360days.


Fig. 6.Brix % at maturity phase in response to chemical ripener application















Sugarcane is known to tolerate temperatures up to 40C. It is
evident that high temperature beyond 40C showed drop in various
metabolic events. The temperature is a main factor which
accelerates invertase activity and inhibits beyond a threshold.
Bhowmik et al., (2001) stated that the increased temperature up to
a certain degree favorably alters the activity, while high
temperatures have a detrimental effect. In the present study also,
similar findings were observed in both varieties. The invertase
activity increased at optimal and near super optimal temperature
followed by a decline when the temperature increased beyond a
threshold.
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